Epitopes in amyloid beta mid-region and conformationally-selective antibodies thereto

ABSTRACT

The disclosure pertains to conformational epitopes in A-beta, antibodies thereto and methods of making and using immunogens and antibodies specific thereto.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/774,707, filed May 9, 2018, which is a national phase entry ofPCT/CA2016/051303, filed Nov. 9, 2016, which claims priority from U.S.Provisional patent application Ser. Nos. 62/253,044 filed Nov. 9, 2015;62/363,566 filed Jul. 18, 2016; 62/365,634 filed Jul. 22, 2016; and62/393,615 filed Sep. 12, 2016; each of these applications beingincorporated herein in their entirety by reference.

INCORPORATION OF SEQUENCE LISTING

A computer readable form of the Sequence Listing“P50442US06_SequenceListing.txt” (8,715 bytes), submitted via EFS-WEBand created on Jul. 13, 2020, is herein incorporated by reference.

FIELD

The present disclosure relates to Amyloid beta (A-beta or Aβ) epitopesand antibodies thereto and more specifically to conformational A-betaepitopes that are predicted and shown to be selectively accessible inA-beta oligomers, as well as related antibody compositions and usesthereof.

BACKGROUND

Amyloid-beta (A-beta), which exists as a 36-43 amino acid peptide, is aproduct released from amyloid precursor protein (APP) by the enzymes βand γ secretase. In AD patients, A-beta can be present in solublemonomers, insoluble fibrils and soluble oligomers. In monomer form,A-beta exists as a predominantly unstructured polypeptide chain. Infibril form, A-beta can aggregate into distinct morphologies, oftenreferred to as strains. Several of these structures have been determinedby solid-state NMR.

For, example, structures for several strains of fibrils are available inthe Protein Data Bank (PDB), a crystallographic database of atomicresolution three dimensional structural data, including a 3-foldsymmetric Aβ structure (PDB entry, 2M4J); a two-fold symmetric structureof Aβ-40 monomers (PDB entry 2LMN), and a single-chain, parallelin-register structure of Aβ-42 monomers (PDB entry 2MXU).

The structure of 2M4J is reported in Lu et al [8], and the structure of2MXU is reported in Xiao et al [9]. The structure of 2LMN is reported inPetkova et al [10].

A-beta oligomers have been shown to kill cell lines and neurons inculture and block a critical synaptic activity that subserves memory,referred to as long term potentiation (LTP), in slice cultures andliving animals.

The structure of the oligomer has not been determined to date. Moreover,NMR and other evidence indicates that the oligomer exists not in asingle well-defined structure, but in a conformationally-plastic,malleable structural ensemble with limited regularity. Moreover, theconcentration of toxic oligomer species is far below either that of themonomer or fibril (estimates vary but are on the order of 1000-foldbelow or more), making this target elusive.

Antibodies that bind A-beta have been described.

WO2009048538A2 titled USE OF ANTI-AMYLOID ANTIBODY IN OCULAR DISEASESdiscloses chimeric antibodies that recognize one or more binding siteson A-beta and are useful for the treatment for ocular diseases.

U.S. Pat. No. 9,221,812B2 titled COMPOUNDS FOR THE TREATMENT OF DISEASESASSOCIATED WITH AMYLOID OR AMYLOID-LIKE PROTEINS describespharmaceutical compositions and discontinuous antibodies that bindA-beta including an epitope between amino acid residues 12 to 24 for thetreatment of amyloid-related diseases.

WO2003070760A2 titled ANTI-AMYLOID BETA ANTIBODIES AND THEIR USEdiscloses antibodies that recognize an A-beta discontinuous epitope,wherein the first region comprises the amino acid sequence AEFRHDSGY(SEQ ID NO: 35) or a fragment thereof and wherein the second regioncomprises the amino acid sequence VHHQKLVFFAEDVG (SEQ ID NO: 33) or afragment thereof.

US20110171243A1 titled COMPOUNDS TREATING AMYLOIDOSES discloses apeptide mimotope capable of inducing the in vivo formation of antibodiesthat bind HQKLVF and/or HQKLVFFAED (SEQ ID NO: 16), and its use.

WO2008088983A1 and WO2001062801A2 disclose a pegylated antibody fragmentthat binds A-beta amino acids 13-28 (HHQKLVFFAEDVGSNK) (SEQ ID NO: 19)and its use in treating A-beta related diseases.

WO2009149487A2 titled COMPOUNDS FOR TREATING SYMPTOMS ASSOCIATED WITHPARKINSON'S DISEASE describes compounds comprising a peptide havingbinding capacity for an antibody specific for an A-beta epitope such asEVHHQKL (SEQ ID NO: 34), HQKLVF (SEQ ID NO: 14) and HQKLVFFAED (SEQ IDNO: 16).

The HHQK (SEQ ID NO: 1) domain is described as involved in plaqueinduction of neurotoxicity in human microglia, as described in Giulian Det al. [11] and Winkler et al. [12]. Non-antibody therapeutic agentsthat bind HHQK (SEQ ID NO: 1) have been disclosed for the treatment ofprotein folding diseases (US20150105344A1, WO2006125324A1).

Antibodies that preferentially or selectively bind A-beta oligomers overmonomers or over fibrils or over both monomers and fibrils aredesirable.

SUMMARY

Described herein are conformational epitopes in A-beta comprising and/orconsisting of residues HHQK (SEQ ID NO: 1) or a part thereof, andantibodies thereto. The epitope is identified as an epitope that may beselectively exposed in the oligomeric species of A-beta, in aconformation that distinguishes it from that in the monomer.

An aspect includes a cyclic compound comprising: an A-beta peptide thepeptide comprising HQK and up to 6 A-beta contiguous residues, and alinker, wherein the linker is covalently coupled to the A-beta peptideN-terminus residue and the A-beta C-terminus residue.

In an embodiment, the A-beta peptide is selected from a peptide having asequence of any one of SEQ ID NOS: 1-16, optionally selected from isselected from HHQK (SEQ ID NO: 1), HQK, HHQKL (SEQ ID NO: 7), VHHQKL(SEQ ID NO: 6), VHHQ (SEQ ID NO: 5), and HQKL (SEQ ID NO: 20).

In another embodiment, the cyclic compound is cyclic peptide.

In another embodiment, the cyclic compound described herein, comprisingi) curvature of Q and/or K in the cyclic compound is at least 10%, atleast 20%, or at least 30% different than the curvature compared to H, Qand/or K in the context of a corresponding linear compound; ii)comprising at least one residue selected from H, Q and K, wherein atleast one dihedral angle of said residue is different by at least 30degrees, at least 40 degrees, at least 50 degrees, at least 60 degrees,at least 70 degrees, at least 80 degrees, at least 90 degrees, at least100 degrees, at least 110 degrees, at least 120 degrees, at least 130degrees, at least 140 degrees, at least 150 degrees, at least 160degrees, at least 170 degrees, at least 180 degrees, at least 190degrees, or at least 200 degrees compared to the corresponding dihedralangle in the context of a corresponding linear compound; iii) the cycliccompound has a conformation for Q and/or K as measured by entropy thatis at least 10%, at least 20%, at least 25%, at least 30%, at least 35%,at least 40% more constrained compared to a corresponding linearcompound; and/or iv) at least H, at least Q, and/or at least K is in amore constrained conformation than the conformation occupied in thelinear peptide comprising HHQK (SEQ ID NO:1) HQK, and/or HQKL (SEQ IDNO: 20).

In another embodiment, the peptide is HHQK (SEQ ID NO: 1) HHQKL (SEQ IDNO: 7) or HQKL (SEQ ID NO: 20).

In another embodiment, the compound further comprises a detectablelabel.

In another embodiment, the linker comprises or consists of 1-8 aminoacids and/or equivalently functioning molecules and/or one or morefunctionalizable moieties.

In another embodiment, the linker amino acids are selected from A and G,and/or wherein the functionalizable moiety is C.

In another embodiment, the linker comprises or consists of amino acidsGCG or CGC.

In another embodiment, the linker comprises a PEG molecule.

In another embodiment, the cyclic compound is selected from thestructures in FIG. 7C.

An aspect includes an immunogen comprising the cyclic compound.

In an embodiment, the compound is coupled to a carrier protein orimmunogenicity enhancing agent.

In another embodiment, the carrier protein is bovine serum albumin (BSA)or the immunogenicity-enhancing agent is keyhole Keyhole LimpetHaemocyanin (KLH).

An aspect includes a composition comprising the compound describedherein or the immunogen described herein.

In an embodiment, the composition described herein, further comprises anadjuvant.

In another embodiment, the adjuvant is aluminum phosphate or aluminumhydroxide.

An aspect includes an isolated conformation specific and/or selectiveantibody that specifically and/or selectively binds to an A-beta peptidehaving a sequence of HQK or a related epitope sequence presented in acyclic compound described herein, optionally having a sequence of SEQ IDNO: 2, 3 or 4.

In an embodiment, the antibody specifically binds an epitope on A-beta,wherein the epitope comprises or consists of at least two consecutiveamino acid residues of HQK predominantly involved in binding to theantibody, wherein the at least two consecutive amino acids are QKembedded within HQK optionally HHQK (SEQ ID NO:1), HQKL (SEQ ID NO:20)or HHQKLV (SEQ ID NO:8), wherein the at least two consecutive aminoacids are HQ embedded within HQK, optionally HHQK (SEQ ID NO:1), HQKL(SEQ ID NO:20), HHQKL (SEQ ID NO: 7), HHQKLV (SEQ ID NO:8), or whereinthe at least two consecutive amino acids are HH embedded within HHQ,optionally HHQK (SEQ ID NO:1) or HHQKLV (SEQ ID NO:8).

In another embodiment, the A-beta peptide and/or epitope comprises orconsists of HHQK (SEQ ID NO:1), VHHQKL (SEQ ID NO:6), VHHQ (SEQ IDNO:5), and HQKL (SEQ ID NO: 20).

In another embodiment, the antibody selectively binds to a cycliccompound comprising HHQK (SEQ ID NO: 1) over a corresponding linearpeptide, optionally wherein the antibody is at least 2 fold, 3 fold, atleast 5 fold, at least 10 fold, at least 20 fold, at least 30 fold, atleast 40 fold, at least 50 fold, at least 100 fold, at least 500 fold,at least 1000 fold more selective for the cyclic compound over thecorresponding linear compound.

In another embodiment, the antibody selectively binds A-beta oligomerover A-beta monomer and/or A-beta fibril.

In another embodiment, the selectivity is at least 2 fold, at least 3fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 30fold, at least 40 fold, at least 50 fold, at least 100 fold, at least500 fold, at least 1000 fold more selective for A-beta oligomer overA-beta monomer and/or A-beta fibril.

In another embodiment, the antibody does not specifically and/orselectively bind a linear peptide comprising sequence HHQK (SEQ IDNO: 1) or a related epitope, optionally wherein the sequence of thelinear peptide is a linear version of a cyclic compound used to raisethe antibody, optionally a linear peptide having a sequence as set forthin SEQ ID NO: 2, 3 or 4.

In another embodiment, the antibody lacks or has negligible binding toA-beta monomer and/or A-beta fibril plaques in situ.

In another embodiment, the antibody is a monoclonal antibody or apolyclonal antibody.

In another embodiment, the antibody is a humanized antibody.

In another embodiment, the antibody is an antibody binding fragmentselected from Fab, Fab′, F(ab′)2, scFv, dsFv, ds-scFv, dimers,nanobodies, minibodies, diabodies, and multimers thereof.

In another embodiment, the antibody described herein comprises a lightchain variable region and a heavy chain variable region, optionallyfused, the heavy chain variable region comprising complementaritydetermining regions CDR-H1, CDR-H2 and CDR-H3, the light chain variableregion comprising complementarity determining region CDR-L1, CDR-L2 andCDR-L3 and with the amino acid sequences of said CDRs comprising thesequences:

CDR-H1 (SEQ ID NO: 22) GYSFTSYW CDR-H2 (SEQ ID NO: 23) VHPGRGVST CDR-H3(SEQ ID NO: 24) SRSHGNTYWFFDV CDR-L1 (SEQ ID NO: 25) QSIVHSNGNTY CDR-L2(SEQ ID NO: 26) KVS CDR-L3 (SEQ ID NO: 27) FQGSHVPFT

In another embodiment, the antibody comprises a heavy chain variableregion comprising: i) an amino acid sequence as set forth in SEQ ID NO:29; ii) an amino acid sequence with at least 50%, at least 60%, at least70%, at least 80%, or at least 90% sequence identity to SEQ ID NO: 29,wherein the CDR sequences are as set forth in SEQ ID NO: 22, 23 and 24,or iii) a conservatively substituted amino acid sequence i).

In another embodiment, the antibody comprises a light chain variableregion comprising i) an amino acid sequence as set forth in SEQ ID NO:31, ii) an amino acid sequence with at least 50%, at least 60%, at least70%, at least 80%, or at least 90% sequence identity to SEQ ID NO: 31,wherein the CDR sequences are as set forth in SEQ ID NO: 25, 26 and 27,or iii) a conservatively substituted amino acid sequence of i).

In another embodiment, the heavy chain variable region amino acidsequence is encoded by a nucleotide sequence as set forth in SEQ ID NO:28 or a codon degenerate or optimized version thereof; and/or theantibody comprises a light chain variable region amino acid sequenceencoded by a nucleotide sequence as set out in SEQ ID NO: 30 or a codondegenerate or optimized version thereof.

In another embodiment, the heavy chain variable region comprises orconsists of an amino acid sequence as set forth in SEQ ID NO: 29 and/orthe light chain variable region comprises or consists of an amino acidsequence as set forth in SEQ ID NO: 31.

In another embodiment, the antibody competes for binding to human A-betawith an antibody comprising the CDR sequences as recited in Table 13.

An aspect includes immunoconjugate comprising the antibody describedherein and a detectable label or cytotoxic agent.

In an embodiment, the detectable label comprises a positron emittingradionuclide, optionally for use in subject imaging such as PET imaging.

An aspect includes a composition comprising the antibody describedherein, or the immunoconjugate described herein, optionally with adiluent.

An aspect includes a nucleic acid molecule encoding a proteinaceousportion of the compound or immunogen described herein, the antibodydescribed herein or proteinaceous immunoconjugates described herein.

An aspect includes a vector comprising the nucleic acid describedherein.

An aspect includes a cell expressing an antibody described herein,optionally wherein the cell is a hybridoma comprising the vectordescribed herein.

An aspect includes a kit comprising the compound described herein, theimmunogen described herein, the antibody described herein, theimmunoconjugate described herein, the composition described herein, thenucleic acid molecule described herein, the vector described herein orthe cell described herein.

An aspect includes a method of making the antibody described herein,comprising administering the compound or immunogen described herein or acomposition comprising said compound or immunogen to a subject andisolating antibody and/or cells expressing antibody specific orselective for the compound or immunogen administered and/or A-betaoligomers, optionally lacking or having negligible binding to a linearpeptide comprising the A-beta peptide and/or lacking or havingnegligible plaque binding.

An aspect includes a method of determining if a biological samplecomprises A-beta, the method comprising:

-   -   a. contacting the biological sample with an antibody described        herein or the immunoconjugate described herein; and    -   b. detecting the presence of any antibody complex.

In an embodiment, the biological sample contains A-beta oligomer themethod comprising:

-   -   a. contacting the sample with the antibody described herein or        the immunoconjugate described herein that is specific and/or        selective for A-beta oligomers under conditions permissive for        forming an antibody: A-beta oligomer complex; and    -   b. detecting the presence of any complex;    -   wherein the presence of detectable complex is indicative that        the sample may contain A-beta oligomer.

In another embodiment, the amount of complex is measured.

In another embodiment, the sample comprises brain tissue or an extractthereof, whole blood, plasma, serum and/or CSF.

In another embodiment, the sample is a human sample.

In another embodiment, the sample is compared to a control, optionally aprevious sample.

In another embodiment, the level of A-beta is detected by SPR.

An aspect includes a method of measuring a level of A-beta in a subject,the method comprising administering to a subject at risk or suspected ofhaving or having AD, an immunoconjugate comprising an antibody describedherein wherein the antibody is conjugated to a detectable label; anddetecting the label, optionally quantitatively detecting the label.

In an embodiment, the label is a positron emitting radionuclide.

An aspect includes a method of inducing an immune response in a subject,comprising administering to the subject a compound or combination ofcompounds described herein, optionally a cyclic compound comprising HQKor HHQK (SEQ ID NO:1) or a related epitope peptide sequence, animmunogen and/or composition comprising said compound or said immunogen;and optionally isolating cells and/or antibodies that specifically orselectively bind the A-beta peptide in the compound or immunogenadministered.

An aspect includes a method of inhibiting A-beta oligomer propagation,the method comprising contacting a cell or tissue expressing A-beta withor administering to a subject in need thereof an effective amount of anA-beta oligomer specific or selective antibody or immunoconjugatedescribed herein, to inhibit A-beta aggregation and/or oligomerpropagation.

An aspect includes a method of treating AD and/or other A-beta amyloidrelated diseases, the method comprising administering to a subject inneed thereof i) an effective amount of an antibody or immunoconjugatedescribed herein, optionally an A-beta oligomer specific or selectiveantibody, or a pharmaceutical composition comprising said antibody; 2)administering an isolated cyclic compound comprising HQK, HHQK (SEQ IDNO:1) or a related epitope sequence or immunogen or pharmaceuticalcomposition comprising said cyclic compound, or 3) a nucleic acid orvector comprising a nucleic acid encoding the antibody of 1 or theimmunogen of 2, to a subject in need thereof.

In an embodiment, a biological sample from the subject to be treated isassessed for the presence or levels of A-beta using an antibodydescribed herein.

In another embodiment, more than one antibody or immunogen isadministered.

In another embodiment, the antibody, immunoconjugate, immunogen,composition or nucleic acid or vector is administered directly to thebrain or other portion of the CNS.

In another embodiment, the composition is a pharmaceutical compositioncomprising the compound or immunogen in admixture with apharmaceutically acceptable, diluent or carrier.

An aspect includes an isolated peptide comprising an A beta peptideconsisting of the sequence of any one of the sequences set forth in thepresent disclosure, optionally Table 15(1).

In an embodiment, the peptide is a cyclic peptide comprising a linkerwherein the linker is covalently coupled to the A-beta peptideN-terminus residue and/or the A-beta C-terminus residue.

In another embodiment, the isolated peptide described herein comprises adetectable label.

An aspect includes a nucleic acid sequence encoding the isolated peptidedescribed herein.

Other features and advantages of the present disclosure will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating preferred embodiments of the disclosure aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the disclosure will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

An embodiment of the present disclosure will now be described inrelation to the drawings in which:

FIG. 1: Likelihood of exposure as a function of sequence, as determinedby the Collective Coordinates method (Panel A) and the Promis Gō method(Panels B, C, D).

FIG. 2: Curvature as a function of residue index. Mean curvature in theequilibrium ensemble for the linear peptide CGHHQKG (SEQ ID NO: 2) isshown (Panel A), along with the curvature for the cyclic peptide (PanelB), and the curvature averaged over both the equilibrium ensemble andthe various monomers in the fibril (Panel C). The convergence checks forthe mean curvature values of all residues in each peptide are shown inPanels D-F. Panel G is a graph shows the converged values of thecurvature for the linear and cyclic peptides along with the curvature inthe fibril. Interestingly, the curvature of Q15 in the cyclic peptide issubstantially lower than that in either the linear peptide or fibril.

FIG. 3: Dihedral angle distributions for the angle C-Cα-Cβ-Cγ (Panel A),C-Cα-N-HN (Panel B), and Cα-Cβ-Cγ-Cδ2 (Panel C), and O-C-Cα-Cβ (Panel D)involving the side chain and backbone atoms of residue 13H. Dihedralangle distributions for residue 14H are shown for angles C-Cα-Cβ-Cγ(Panel E), C-Cα-N-HN (Panel F), and Cα-Cβ-Cγ-Cδ2 (Panel G), andO-C-Cα-Cβ (Panel H). For 15Q, dihedral angle distributions are shown forangles C-Cα-Cβ-Cγ (Panel I), C-Cα-N-HN (Panel J), Nε2-Cγ-Cδ-Cβ (Panel K)and O-C-Cα-Cβ (Panel L). For 16K, dihedral angle distributions are shownfor angles C-Cα-Cβ-Cγ (Panel M), C-Cα-N-HN (Panel N), and O-C-Cα-Cβ(Panel O). The overlapping percentage values are provided in Table 2.The peak values of the dihedral angles for the distributions are givenin Table 3.

FIG. 4: Entropy change of individual dihedral angles in the linear andcyclic peptides relative to the entropy in the fibril, plotted for eachresidue 13H (Panel A), 14H (Panel B), 15Q (Panel C), and 16K (Panel D).Panel E: Difference from the fibril of the non-Ramachandran entropy ofindividual residues—i.e. the backbone Ramachandran entropy is notincluded. Panel F: Side chain plus backbone (total) conformationalentropy of individual residues, minus the corresponding quantity in thefibril. Panel G plots the entropy loss of each residue relative to thelinear peptide, for both the cyclic peptide and fibril.

FIG. 5: Equilibrium backbone Ramachandran angles for residues 13H, 14H,15Q and 16K, in cyclic (left panel) and linear (middle panel) forms ofthe peptide CGHHQKG (SEQ ID NO: 2), along with the backbone Ramachandranangles for the residues in the context of the fibril 2M4J (right panel)in Panel A. The overlap probabilities Ramachandran angles are shown inTable 4. The peak angles of the corresponding distributions are shown inTable 5. Panels B-E show a separate representation of the individualbackbone Ramachandran angles ϕ and ψ for each amino acid.

FIG. 6: Plots of solubility and solvent accessible surface area (SASA),for the residues HHQK (SEQ ID NO: 1). Panel A shows the solubility as afunction of residue index, with HHQK (SEQ ID NO: 1) delineated byvertical dashed lines. Panel B shows the SASA for residues HHQK (SEQ IDNO: 1) where HHQK (SEQ ID NO: 1) in the cyclic peptide is represented asa dotted line, HHQK (SEQ ID NO: 1) in the linear peptide is representedin solid light grey line, and HHQK (SEQ ID NO: 1) in the context of thefibril 2M4J is represented in solid dark grey line. Panel C shows theSASA weighted by the solubility of each residue, as described below.Panel D shows the weighted ΔSASA depicting the difference in SASA ofcyclic and linear peptides with respect to the fibril 2M4J.

FIG. 7: Centroid structures of the cyclic and linear peptide ensemblesof CGHHQKG (SEQ ID NO: 2). The black colored conformation is thecentroid of the largest cluster of the cyclic peptide, and so bestrepresents the typical conformation of the cyclic peptide. The whitecolored conformation is the centroid of the largest cluster of thelinear peptide, and may best represent the typical conformation of thelinear peptide. The linear centroid is aligned to the cyclic centroid.The superimposed aligned structures show that different dihedral anglesand overall epitope conformations tend to be preferred for the linearand cyclic peptides. Panel A: Aligned centroid structures of residues13H, 14H, 15Q, and 16K (HHQK SEQ ID NO: 1) in cyclic and linear peptidesare shown in overlapping pictures from two different viewpoints. PanelB: Three views of the cyclic peptide structure CGHHQKG (SEQ ID NO: 2),and linear peptide structure CGHHQKG (SEQ ID NO: 2), both rendered inlicorice representation so the orientations of the side chains can beseen. Panel C: Schematic representations of cyclic peptides containingthe epitope residues HHQK (SEQ ID NO: 1), including the cyclic peptideCGHHQKG (SEQ ID NO: 2) with circular peptide bond, the cyclic peptideC-PEG2-HHQKG (SEQ ID NO: 3) with PEG2 linker between the C and Hresidues, and the cyclic peptide CGHHQK-PEG2 (SEQ ID NO: 4) with PEG2linker between the K and C residues.

FIG. 8: The solvent-accessible surface area (SASA) of the epitope HHQK(SEQ ID NO:1) is shown in the context of the linear and the cyclicpeptides of sequence CGHHQKG (SEQ ID NO:2), and the correspondingportion of A-beta40 polypeptide 2M4J. Panel A shows the SASA of theepitope HHQK (SEQ ID NO:1) for the linear (left) and the cyclic (right)peptide separately. Panel B shows the cyclic and the fibril SASAsaligned (left), as well as the SASAs of the aligned cyclic and linearpeptides. Both panels show that the antigenic surface presented by thecyclic peptide is distinct from either the linear or fibril. Panel Cshows the epitope HHQK (SEQ ID NO:1) sequence within the A-beta40 fibril2M4J, showing only the atoms with solvent exposure. The surface area ispresented differently in the cyclic and linear peptides. This indicatesthat antibodies may be selected to have high affinity to cyclic HHQK(SEQ ID NO:1) compounds and low affinity to A-beta40 polypeptidemonomers.

FIG. 9: Clustering plots by root mean squared deviation (RMSD); axescorrespond to the RMSD of HHQK (SEQ ID NO:1) relative to HHQK (SEQ IDNO:1) in the centroid structure of the cyclic peptide ensemble, the RMSDof HHQK (SEQ ID NO:1) to HHQK (SEQ ID NO: 1) in the centroid structureof the linear peptide ensemble, and the RMSD of HHQK (SEQ ID NO:1) toHHQK (SEQ ID NO:1) in the centroid structure of the fibril ensemble ofPDB ID 2M4J. Each point corresponds to a given conformation taken fromeither the cyclic peptide equilibrium ensemble (circles as noted in thelegend), the linear peptide equilibrium ensemble (+ symbols as noted inthe legend), or the fibril equilibrium ensemble starting from PDB ID2M4J (inverted triangles as noted in the legend). Three differentviewpoints are presented in Panels A-C. The cyclic peptide ensemble,shown as dark gray circles, shows conformational distinction from eitherthe linear or the fibril ensemble, which may be quantified by computingthe overlap percentages between the distributions as shown in panelsD-H. Panels D-I show convergence checks of the overlap between thedistributions of the cyclic, linear and fibril forms of the peptide.Panel J examines the effects of single residue deletions on thestructural overlap of the linear ensemble with the 90% cyclic ensemble.If a single amino acid confers conformational selectivity, then removingit from the structural alignment will result in a significantly higheroverlap. By this test, K16 may confer the most conformationalselectivity to the cyclic peptide.

FIG. 10: Clustering plots by RMSD for other fibril strain conformations;axes correspond to the RMSD of HHQK (SEQ ID NO:1) relative to HHQK (SEQID NO:1) in the centroid structure of the cyclic peptide ensemble, theRMSD of HHQK (SEQ ID NO:1) to HHQK (SEQ ID NO:1) in the centroidstructure of the linear peptide ensemble, and the RMSD of HHQK (SEQ IDNO:1) to HHQK (SEQ ID NO:1) in the centroid structure of the equilibriumensembles for several fibril models of A-beta. Each point corresponds toa given conformation taken from either the cyclic peptide, or various“strains” of fibril equilibrium ensembles, from PDB IDs 2MXU (Panel A, 2views), 2LMP (Panel B), and 2LMN (Panel C, 2 views).

FIG. 11: Surface plasmon resonance (SPR) binding assay of tissue culturesupernatant clones to cyclic peptide and linear peptide in Panel A, andA-beta oligomer and A-beta monomer in Panel B.

FIG. 12: Plot comparing tissue culture supernatant clones binding in SPRbinding assay versus ELISA.

FIG. 13: SPR binding assay of select clones to cyclic peptide (circles),linear peptide (squares), A-beta monomer (upward triangle), and A-betaoligomer (downward triangle). Asterisk indicates a clone reactive tounstructured linear peptide for control purposes.

FIG. 14: Immunohistochemical staining of plaque from cadaveric AD brainusing 6E10 positive control antibody (A) and a selected and purifiedmonoclonal antibody (301-17, 12G11) raised against cyclo(CGHHQKG) (SEQID NO: 2) (B).

FIG. 15: Secondary screening of selected and purified antibodies usingan SPR indirect (capture) binding assay. SPR binding response of A-betaoligomer to captured antibody minus binding response of A-beta monomerto captured antibody (circle); SPR binding response of pooled solublebrain extract from AD patients to captured antibody minus bindingresponse of pooled brain extract from non-AD controls to capturedantibody (triangle); SPR binding response of pooled cerebrospinal fluid(CSF) from AD patients to captured antibody minus binding response ofpooled CSF from non-AD controls to captured antibody (square).

FIG. 16: Verification of Antibody binding to stable A-beta oligomers.SPR sensorgrams and binding response plots of varying concentrations ofcommercially-prepared stable A-beta oligomers binding to immobilizedantibodies. Panel A shows results with the positive control mAb6E10,Panel B with the negative isotype control and Panel C with antibodyraised against cyclo (CGHHQKG) (SEQ ID NO: 2). Panel D plots binding ofseveral antibody clones raised against cyclic peptide comprising HHQK(SEQ ID No: 1), with A-beta oligomer at a concentration of 1 micromolar.

FIG. 17: A plot showing propagation of A-beta aggregation in vitro inthe presence or absence of representative antibody raised using a cyclicpeptide comprising HHQK (SEQ ID NO: 1).

FIG. 18: A plot showing the viability of rat primary cortical neuronsexposed to toxic A-beta oligomers (AβO) in the presence or absence ofdifferent molar ratios of a negative isotype control (A) or an antibodyraised using a cyclic peptide comprising HHQK (SEQ ID NO:1) (B).Controls include neurons cultured alone (CTRL), neurons incubated withantibody without oligomers and neurons cultured with the neuroprotectivehumanin peptide (HNG) with or without oligomers.

Table 1 shows the curvature value by residue of 13H, 14H, 15Q, and 16Kin linear, cyclic and fibril 2M4J forms.

Table 2 shows the overlapping percentages of distribution in dihedralangles presented in FIG. 3.

Table 3 shows the peak values of the dihedral angle distribution forthose dihedral angles whose distributions show differences between thecyclic peptide and other species. Column 1 is the specific dihedralconsidered, column 2 is the peak value of the dihedral distribution forthat angle in the context of the cyclic peptide CGHHQKG (SEQ ID NO: 2),column 3 is the peak value of the dihedral distribution for that anglein the context of the linear peptide CGHHQKG (SEQ ID NO: 2), column 4 isthe peak value of the dihedral distribution for the peptide HHQK (SEQ IDNO: 1) in the context of the fibril structure 2M4J, and column 5 is thedifference of the peak values of the dihedral distributions between thelinear and cyclic peptides. See also FIG. 3.

Table 4 shows the overlap probabilities of Ramachandran angles of theresidues 13H, 14H, 15Q, and 16K presented in FIG. 5. Specifically, thefraction of the linear ensemble that adopts conformations consistentwith the cyclic peptide is 76%, 10%, 10%, 32% for H13, H14, Q15 and K16respectively. This indicates for example that H14 and Q15 in the freepeptide rarely adopt cyclic-like conformations.

Table 5 shows peak values of the Ramachandran backbone phi/psi angledistributions of the residues 13H, 14H, 15Q, and 16K. The first columnis the residue considered, which manifests two angles, phi and psi,indicated in parenthesis. The 2^(nd) column indicates the peak values ofthe Ramachandran phi/psi angles for each residue in the context of thelinear peptide CGHHQKG (SEQ ID NO: 2), while the 3^(rd) column indicatesthe peak values of the Ramachandran phi/psi angles for each residue inthe context of the cyclic peptide CGHHQKG (SEQ ID NO: 2), and the lastcolumn indicates the peak values of the Ramachandran phi/psi angles foreach residue in the context of the fibril structure 2M4J. See FIG. 5.

Table 6 shows the overlapping percentage of the RMSD clustering betweenthe linear, cyclic and fibril (2M4J) forms of the peptide as presentedin FIG. 9.

Table 7 gives the values of the backbone and sidechain dihedral anglesfor residues 13H, 14H, 15Q, and 16K, in the centroid conformations ofthe cyclic, linear, and fibril ensembles.

Table 8 shows the binding properties of selected tissue culturesupernatant clones.

Table 9 shows the binding properties summary for selected antibodies.

Table 10 lists the oligomer binding—monomer binding for an antibodyraised against cyclo(CGHHQKG) (SEQ ID NO:2).

Table 11 lists properties of antibodies tested on formalin fixedtissues.

Table 12 is an exemplary toxicity assay

Table 13 lists CDR sequences.

Table 14 lists heavy chain and light chain variable sequences.

Table 15 is a table of A-beta sequences.

Table 16 lists A-beta full length sequence.

DETAILED DESCRIPTION OF THE DISCLOSURE

Provided herein are antibodies, immunotherapeutic compositions andmethods which may target epitopes preferentially accessible in toxicoligomeric species of A-beta, including oligomeric species associatedwith Alzheimer's disease. A region in A-beta has been identified thatmay be specifically and/or selectively accessible to antibody binding inoligomeric species of A-beta.

As demonstrated herein, generation of oligomer-specific or oligomerselective antibodies was accomplished through the identification oftargets on A-beta peptide that are not present, or present to a lesserdegree, on either the monomer and/or fibril. Oligomer-specific epitopesneed not differ in primary sequence from the corresponding segment inthe monomer or fibril, however they would be conformationally distinctin the context of the oligomer. That is, they would present a distinctconformation in terms of backbone and/or side-chain orientation in theoligomer that would not be present (or would be unfavourable) in themonomer and/or fibril.

Antibodies raised to linear peptide regions tend not to be selective foroligomer, and thus bind to monomer as well.

As described herein, to develop antibodies that may be selective foroligomeric forms of A-beta, the inventors sought to identify regions ofA-beta sequence that are prone to disruption in the context of thefibril, and that may be exposed as well on the surface of the oligomer.

As described the Examples, the inventors have identified a regionpredicted to be prone to disruption in the context of the fibril. Theinventors designed cyclic compounds comprising the identified targetregion to satisfy criteria of an alternate conformation such as adifferent curvature profile vs residue index, higher exposed surfacearea, and/or did not readily align by root mean squared deviation (RMSD)to either the linear or fibril ensembles.

As shown in the Examples, an immunogen comprising the cyclic compound ofSEQ ID NO: 2 was used to produce monoclonal antibodies. As shown in theExamples, antibodies could be raised using a cyclic peptide comprisingthe target region, that selectively bound the cyclic peptide compared toa linear peptide of the same sequence (e.g. corresponding linearsequence). Experimental results are described and identifyepitope-specific and conformationally selective antibodies that bindsynthetic oligomer selectively compared to synthetic monomers, bind CSFfrom AD patients preferentially over control CSF and/or bind solublebrain extract from AD patients preferentially over control soluble brainextract. Further staining of AD brain tissue identified antibodies thatshow no or negligible plaque binding and in vitro studies found that theantibodies inhibited Aβ oligomer propagation and aggregation.

I. Definitions

As used herein, the term ‘A-beta’ may alternately be referred to as‘amyloid beta’, ‘amyloid β’, A-beta, A-beta or ‘Aβ’. Amyloid beta is apeptide of 36-43 amino acids and includes all wild-type and mutant formsof all species, particularly human A-beta. A-beta40 refers to the 40amino acid form; A-beta42 refers to the 42 amino acid form, etc. Theamino acid sequence of human wildtype A-beta42 is shown in SEQ ID NO:32.

As used herein, the term “A-beta monomer” herein refers to any of theindividual subunit forms of the A-beta (e.g. 1-40, 1-42, 1-43) peptide.

As used herein, the term “A-beta oligomer” herein refers to a pluralityof any of the A-beta subunits wherein several (e.g. at least two) A-betamonomers are non-covalently aggregated in a conformationally-flexible,partially-ordered, three-dimensional globule of less than about 100, ormore typically less than about 50 monomers. For example, an oligomer maycontain 3 or 4 or 5 or more monomers. The term “A-beta oligomer” as usedherein includes both synthetic A-beta oligomer and/or native A-betaoligomer. “Native A-beta oligomer” refers to A-beta oligomer formed invivo, for example in the brain and CSF of a subject with AD.

As used herein, the term “A-beta fibril” refers to a molecular structurethat comprises assemblies of non-covalently associated, individualA-beta peptides which show fibrillary structure under an electronmicroscope. The fibrillary structure is typically a “cross beta”structure; there is no theoretical upper limit on the size of multimers,and fibrils may comprise thousands or many thousands of monomers.Fibrils can aggregate by the thousands to form senile plaques, one ofthe primary pathological morphologies diagnostic of AD.

The term “HHQK” means the amino acid sequence histidine, histidine,glutamine, lysine, as shown in SEQ ID NO: 1. Similarly HQK, HHQ, VHHQ(SEQ ID NO:5), VHHQKL (SEQ ID NO:6), HHQKL (SEQ ID NO: 7), HHQKLV (SEQID NO: 8), HQKL (SEQ ID NO: 20) refer to the amino acid sequenceidentified by the 1-letter amino acid code. Depending on the context,the reference of the amino acid sequence can refer to a sequence inA-beta or an isolated peptide, such as the amino acid sequence of acyclic compound.

The term “alternate conformation than occupied by 13H, 14H, 15Q and/or16K in the linear compound, monomer and/or fibril” as used herein meanshaving one or more differing conformational properties selected fromsolvent accessibility, entropy, curvature (e.g. in the context of apeptide comprising HHQK (SEQ ID NO: 1) as measured for example in thecyclic peptide described in the examples, RMSD structural alignment, anddihedral angle of one or more backbone or side chain dihedral anglescompared to said property for 13H, 14H, 15Q and/or 16K in acorresponding A-beta linear peptide, A-beta monomer and/or A-beta fibrilstructures as shown for example in PDBs 2M4J, and shown in FIGS. 1-10and/or in the Tables. Further, the term “alternate conformation thanoccupied by 15Q and/or 16K in the linear peptide” as used herein meanshaving one or more differing conformational properties selected fromsolvent accessibility, entropy, curvature (e.g. in the context of apeptide comprising HHQK (SEQ ID NO:1) as measured for example in thecyclic peptide described in the examples), RMSD structural alignment,and dihedral angle of one or more backbone or side chain dihedral anglescompared to said property for 15Q and/or 16K in the corresponding linearA-beta peptide or HHQK (SEQ ID NO:1). A different curvature profile ofthe epitope in the cyclic peptide ensemble than either the linear orfibril ensembles implies that conformational selectivity may beconferred, particularly by residue Q15, which exhibits substantiallydifferent curvature in the cyclic peptide than either the linear peptideor fibril, according to FIG. 2. Residue K16 also exhibits substantiallydifferent curvature for the cyclic peptide than for the linear peptide,adopting a curvature more similar to the fibril. The curvature in thefibril is clearly reduced from that and either the cyclic or linearpeptides: HHQK (SEQ ID NO:1) is relatively extended in the fibril.According to FIG. 3, for residue 13H, dihedrals C-CA-N-HN and O-C-CA-CBdistinguish both linear and cyclic peptides of HHQK (SEQ ID NO:1) fromthe corresponding dihedral angles in the fibril. For residue 14H,dihedral angles C-CA-N-HN and O-C-CA-CB distinguish the cyclic dihedralangle distribution from the corresponding distributions in either thelinear or fibril ensembles. Likewise, for residue 15Q, dihedral anglesC-CA-N-HN and O-C-CA-CB distinguish the cyclic dihedral angledistribution from the corresponding distributions in either the linearor fibril ensembles. For residue 16K, dihedral angle O-C-CA-CBdistinguishes the cyclic peptide from either the linear or fibrilensembles, and dihedral angle C-CA-N-HN distinguishes both cyclic andlinear peptides from the fibril. According to FIG. 5B, the backboneRamachandran angles ϕ and ψ of 13H distinguish the linear and cyclicpeptides from the fibril, but not from each other. For 14H, FIG. 5Cshows that Ramachandran angles ϕ and ψ of the cyclic peptide are bothdistinct from either the linear or fibril ensembles. Likewise for 15Qand 16K, FIGS. 5D and E show that the Ramachandran angles ϕ and ψ of thecyclic peptide are distinct from those in either the linear or fibrilensembles. FIGS. 4F, G demonstrate that the cyclic peptide is moreconstrained than the linear peptide, but less constrained than thefibril. FIG. 4F shows that 15Q and 16K are more constrained in thecyclic peptide ensemble then they are in the linear peptide, suggesting,together with the dihedral angle differences described above, that themonomer will only rarely populate conformations consistent with thecyclic peptide. Despite being more constrained in the cyclic ensemblethan in the linear ensemble, the cyclic peptide is somewhat more solventexposed than the linear peptide (FIG. 6B), thus revealing more antigenicsurface. By direct structural alignment (FIGS. 7, 8, 9), the cyclicpeptide reveals a structural ensemble that is distinct from either thelinear peptide or from HHQK (SEQ ID NO: 1) in the context of the fibril.

The term “amino acid” includes all of the naturally occurring aminoacids as well as modified L-amino acids. The atoms of the amino acid caninclude different isotopes. For example, the amino acids can comprisedeuterium substituted for hydrogen nitrogen-15 substituted fornitrogen-14, and carbon-13 substituted for carbon-12 and other similarchanges.

The term “antibody” as used herein is intended to include, monoclonalantibodies, polyclonal antibodies, single chain, veneered, humanized andother chimeric antibodies and binding fragments thereof, including forexample a single chain Fab fragment, Fab′2 fragment or single chain Fvfragment. The antibody may be from recombinant sources and/or producedin animals such as rabbits, llamas, sharks etc. Also included are humanantibodies that can be produced in transgenic animals or usingbiochemical techniques or can be isolated from a library such as a phagelibrary. Humanized or other chimeric antibodies may include sequencesfrom one or more than one isotype or class or species.

The phrase “isolated antibody” refers to antibody produced in vivo or invitro that has been removed from the source that produced the antibody,for example, an animal, hybridoma or other cell line (such asrecombinant insect, yeast or bacteria cells that produce antibody). Theisolated antibody is optionally “purified”, which means at least: 80%,85%, 90%, 95%, 98% or 99% purity.

The term “binding fragment” as used herein to a part or portion of anantibody or antibody chain comprising fewer amino acid residues than anintact or complete antibody or antibody chain and which binds theantigen or competes with intact antibody. Exemplary binding fragmentsinclude without limitations Fab, Fab′, F(ab′)2, scFv, dsFv, ds-scFv,dimers, nanobodies, minibodies, diabodies, and multimers thereof.Fragments can be obtained via chemical or enzymatic treatment of anintact or complete antibody or antibody chain. Fragments can also beobtained by recombinant means. For example, F(ab′)2 fragments can begenerated by treating the antibody with pepsin. The resulting F(ab′)2fragment can be treated to reduce disulfide bridges to produce Fab′fragments. Papain digestion can lead to the formation of Fab fragments.Fab, Fab′ and F(ab′)2, scFv, dsFv, ds-scFv, dimers, minibodies,diabodies, bispecific antibody fragments and other fragments can also beconstructed by recombinant expression techniques.

The terms “IMGT numbering” or “ImMunoGeneTics database numbering”, whichare recognized in the art, refer to a system of numbering amino acidresidues which are more variable (i.e. hypervariable) than other aminoacid residues in the heavy and light chain variable regions of anantibody, or antigen binding portion thereof.

When an antibody is said to bind to an epitope within specifiedresidues, such as HHQK (SEQ ID NO: 1), what is meant is that theantibody specifically binds to a peptide or polypeptide containing thespecified residues or a part thereof for example at least 1 residue orat least 2 residues, with a minimum affinity, and does not bind anunrelated sequence or unrelated sequence spatial orientation greaterthan for example an isotype control antibody. Such an antibody does notnecessarily contact each residue of HHQK (SEQ ID NO:1) (or a relatedepitope), and every single amino acid substitution or deletion withinsaid epitope does not necessarily significantly affect and/or equallyaffect binding affinity.

When an antibody is said to selectively bind an epitope such as aconformational epitope, such as HHQK (SEQ ID NO: 1), what is meant isthat the antibody preferentially binds one or more particularconformations containing the specified residues or a part thereof withgreater affinity than it binds said residues in another conformation.For example, when an antibody is said to selectively bind a cyclopeptidecomprising HHQK or related epitope relative to a corresponding linearpeptide, the antibody binds the cyclopeptide with at least a 2 foldgreater affinity than it binds the linear peptide.

As used herein, the term “conformational epitope” refers to an epitopewhere the epitope amino acid sequence has a particular three-dimensionalstructure wherein at least an aspect of the three-dimensional structurenot present or less likely to be present in a corresponding linearpeptide is specifically and/or selectively recognized by the cognateantibody. The epitope e.g. HHQK (SEQ ID NO: 1) may be partially orcompletely exposed on the molecular surface of oligomeric A-beta andpartially or completely obscured from antibody recognition in monomericor fibrillar plaque A-beta. Antibodies which specifically bind aconformation-specific epitope recognize the spatial arrangement of oneor more of the amino acids of that conformation-specific epitope. Forexample, an HHQK (SEQ ID NO:1) conformational epitope refers to anepitope of HHQK (SEQ ID NO:1) that is recognized by antibodiesselectively, for example at least 2 fold, 3 fold, 5 fold, 10 fold, 50fold, 100 fold, 250 fold, 500 fold or 1000 fold or greater moreselectivity as compared to antibodies raised using linear HHQK (SEQ IDNO:1).

The term “related epitope” as used herein means at least two residues ofHHQK (SEQ ID NO:1) that are antigenic, optionally sequences comprisingHQK, and/or sequences comprising 1 2 or 3 amino acid residues in aA-beta N-terminal and/or 1 residue C-terminal to at least two residuesof HHQK (SEQ ID NO: 1). For example it is shown herein HHQK (SEQ IDNO:1) and HQKL (SEQ ID NO: 20) which share the subregion HQK wereidentified as regions prone to disorder in an A-beta fibril. HQK andHQKL are accordingly related epitopes. Exemplary related epitopes caninclude epitopes whose sequences are shown in Table 15 (1). The relatedepitope is for example up to 6 A-beta residues.

The term “constrained conformation” as used herein with respect to anamino acid or a side chain thereof, within a sequence of amino acids(e.g. 13H, 14H, 15Q and/or 16K in HHQK (SEQ ID NO:1)), or with respectto a sequence of amino acids in a larger polypeptide, means decreasedrotational mobility of the amino acid dihedral angles, relative to acorresponding linear peptide sequence, or the sequence in the context ofthe larger polypeptide, resulting in a decrease in the number ofpermissible conformations. This can be quantified for example by findingthe entropy reduction for the ensemble of backbone and side chaindihedral angle degrees of freedom, and is plotted in FIG. 4G for eachamino acid, for the entropy reduction in the cyclic ensemble and fibrilensemble relative to the linear ensemble. For example, if the sidechains in the sequence have less conformational freedom than the linearpeptide, the entropy will be reduced. The entropy increase from thefibril ensemble, for both the linear and cyclic peptide ensembles, isplotted in FIGS. 4A-D for the individual independent dihedral angles ineach amino acid. The entropy increase from the fibril ensemble, for boththe linear and cyclic peptide ensembles, is plotted in FIG. 4F for eachamino acid in HHQK (SEQ ID NO: 1). Conformational restriction from thelinear peptide would enhance the conformational selectivity ofantibodies specifically raised to this antigen. The amino acid sequenceHHQK (SEQ ID NO: 1) is most constrained in the fibril structure, whereit has less conformational freedom than either the cyclic peptide or themonomer; it is also more in constrained in the cyclic peptide ensemblethen it is in the linear peptide ensemble. FIG. 4F shows that Q15 andK16 (and to a lesser extent H13) have less entropy in the cyclic peptideensemble than they do in the equilibrium linear peptide ensemble, butthat they have more entropy in the cyclic peptide ensemble then they doin the equilibrium fibril ensemble. The term “more constrainedconformation” as used herein also means that the dihedral angledistribution (ensemble of allowable dihedral angles) of one or moredihedral angles is at least 10% more constrained than in the comparatorconformation, as determined for example by the entropy of the aminoacids, for example H, Q and/or K (e.g. a more constrained conformationhas lower entropy). Specifically, the percent reduction in entropy asmeasured by the average entropy change relative to the larger of theentropy of the linear and cyclic peptides,[|ΔS(cyclic)−ΔS(linear)|/(max(|ΔS(cyclic)|,|ΔS(linear)|)], of HHQK (SEQID NO:1) in the overall more constrained cyclic conformational ensembleis on average reduced by more than 10% or reduced by more than 20% orreduced by more than 30% or reduced by more than 40%, from theunconstrained conformational ensemble. The entropy ΔS in the aboveformula is obtained as the entropy relative to the fibril, e.g.ΔS(cyclic)=S(cyclic)−S(fibril). Specifically, the percent reduction inentropy according to the data plotted in FIG. 4F, is 85%, 65%, 53%, and43% for residues H, H, Q, and K respectively. The overall entropydifference of the linear to the cyclic peptide (relative to the fibrilentropy) ismean[|ΔS(cyclic)−ΔS(linear)|/(max(|ΔS(cyclic)|,|ΔS(linear)|)]=61%.

The term “no or negligible plaque binding” or “lacks or has negligibleplaque binding” as used herein with respect to an antibody means thatthe antibody does not show typical plaque morphology staining onimmunohistochemistry (e.g. in situ) and the level of staining iscomparable to or no more than 2 fold the level seen with an IgG negative(e.g. irrelevant) isotype control.

The term “Isolated peptide” refers to peptide that has been produced,for example, by recombinant or synthetic techniques, and removed fromthe source that produced the peptide, such as recombinant cells orresidual peptide synthesis reactants. The isolated peptide is optionally“purified”, which means at least: 80%, 85%, 90%, 95%, 98% or 99% purityand optionally pharmaceutical grade purity.

The term “detectable label” as used herein refers to moieties such aspeptide sequences (such a myc tag, HA-tag, V5-tag or NE-tag),fluorescent proteins that can be appended or introduced into a peptideor compound described herein and which is capable of producing, eitherdirectly or indirectly, a detectable signal. For example, the label maybe radio-opaque, positron-emitting radionuclide (for example for use inPET imaging), or a radioisotope, such as ³H, ¹³N, ¹⁴C, ¹⁸F, ³²P, ³⁵S,¹²³I, ¹²⁵I, ¹³¹I; a fluorescent (fluorophore) or chemiluminescent(chromophore) compound, such as fluorescein isothiocyanate, rhodamine orluciferin; an enzyme, such as alkaline phosphatase, beta-galactosidaseor horseradish peroxidase; an imaging agent; or a metal ion. Thedetectable label may be also detectable indirectly for example usingsecondary antibody.

The term “epitope” as commonly used means an antibody binding site,typically a polypeptide segment, in an antigen that is specificallyrecognized by the antibody. As used herein “epitope” can also refer tothe amino acid sequences or part thereof identified on A-beta using thecollective coordinates method described. For example an antibodygenerated against an isolated peptide corresponding to a cyclic compoundcomprising the identified target region HHQK SEQ ID NO:1), recognizespart or all of said epitope sequence. An epitope is “accessible” in thecontext of the present specification when it is accessible to binding byan antibody.

The term “greater affinity” as used herein refers to a relative degreeof antibody binding where an antibody X binds to target Y more strongly(K_(on)) and/or with a smaller dissociation constant (K_(off)) than totarget Z, and in this context antibody X has a greater affinity fortarget Y than for Z. Likewise, the term “lesser affinity” herein refersto a degree of antibody binding where an antibody X binds to target Yless strongly and/or with a larger dissociation constant than to targetZ, and in this context antibody X has a lesser affinity for target Ythan for Z. The affinity of binding between an antibody and its targetantigen, can be expressed as K_(A) equal to 1/K_(D) where K_(D) is equalto k_(on)/k_(off). The k_(on) and k_(off) values can be measured usingsurface plasmon resonance technology, for example using a MolecularAffinity Screening System (MASS-1) (Sierra Sensors GmbH, Hamburg,Germany). An antibody that is selective for a conformation presented ina cyclic compound optional a cyclic peptide for example has a greateraffinity for the cyclic compound (e.g. cyclic peptide) compared to acorresponding sequence in linear form (e.g. the sequence non-cyclized).

Also as used herein, the term “immunogenic” refers to substances thatelicit the production of antibodies, activate T-cells and other reactiveimmune cells directed against an antigenic portion of the immunogen.

The term “corresponding linear compound” with regard to a cycliccompound refers to a compound, optionally a peptide, comprising orconsisting of the same sequence or chemical moieties as the cycliccompound but in linear (i.e. non-cyclized) form, for example havingproperties as would be present in solution of a linear peptide. Forexample, the corresponding linear compound can be the synthesizedpeptide that is not cyclized.

As used herein “specifically binds” in reference to an antibody meansthat the antibody recognizes an epitope sequence and binds to its targetantigen with a minimum affinity. For example a multivalent antibodybinds its target with a K_(D) of at least 1e−6, at least 1e−7, at least1e−8, at least 1e−9, or at least 1e−10. Affinities greater than at least1e−8 may be preferred. An antigen binding fragment such as Fab fragmentcomprising one variable domain, may bind its target with a 10 fold or100 fold less affinity than a multivalent interaction with anon-fragmented antibody.

The term “selectively binds” as used herein with respect to an antibodythat selectively binds a form of A-beta (e.g. fibril, monomer oroligomer) or a cyclic compound means that the antibody binds the formwith at least 2 fold, at least 3 fold, or at least 5 fold, at least 10fold, at least 100 fold, at least 250 fold, at least 500 fold or atleast 1000 fold or more greater affinity. Accordingly an antibody thatis more selective for a particular conformation (e.g. oligomer)preferentially binds the particular form of A-beta with at least 2 foldetc. greater affinity compared to another form and/or a linear peptide.

The term “linker” as used herein means a chemical moiety that can becovalently linked to the peptide comprising HHQK (SEQ ID NO:1) epitopepeptide, optionally linked to HHQK (SEQ ID NO:1) peptide N- andC-termini to produce a cyclic compound. The linker can comprise a spacerand/or one or more functionalizable moieties. The linker can be linkedvia the functionalizable moieties to a carrier protein or an immunogenenhancing agent such as keyhole limpet hemocyanin (KLH).

The term “spacer” as used herein means any preferably non-immunogenic orpoorly immunogenic chemical moiety that can be covalently-linkeddirectly or indirectly to a peptide N- and C-termini to produce a cycliccompound of longer length than the peptide itself, for example thespacer can be linked to the N- and C-termini of a peptide consisting ofHHQK (SEQ ID NO:1) to produce a cyclic compound of longer backbonelength than the HHQK (SEQ ID NO:1) sequence itself. That is, whencyclized the peptide with a spacer (for example of 3 amino acidresidues) makes a larger closed circle than the peptide without aspacer. The spacer may include, but is not limited to, non-immunogenicmoieties such as G, A, or PEG repeats, e.g. when in combination with thepeptide being GHHQKG (SEQ ID NO: 9) HHQKG (SEQ ID NO: 10), GHHQK (SEQ IDNO: 11), etc. The spacer may comprise or be coupled to one or morefunctionalizing moieties, such as one or more cysteine (C) residues,which can be interspersed within the spacer or covalently linked to oneor both ends of the spacer. Where a functionalizable moiety such as a Cresidue is covalently linked to one or more termini of the spacer, thespacer is indirectly covalently linked to the peptide. The spacer canalso comprise the functionalizable moiety in a spacer residue as in thecase where a biotin molecule is introduced into an amino acid residue.

The term “functionalizable moiety” as used herein refers to a chemicalentity with a “functional group” which as used herein refers to a groupof atoms or a single atom that will react with another group of atoms ora single atom (so called “complementary functional group”) to form achemical interaction between the two groups or atoms. In the case ofcysteine, the functional group can be —SH which can be reacted to form adisulfide bond. Accordingly the linker can for example be CCC. Thereaction with another group of atoms can be covalent or a strongnon-covalent bond, for example as in the case as biotin-streptavidinbonds, which can have Kd˜1e−14. A strong non-covalent bond as usedherein means an interaction with a Kd of at least 1e−9, at least 1e−10,at least 1e−11, at least 1e−12, at least 1e−13 or at least 1e−14.

Proteins and/or other agents may be functionalized (e.g. coupled) to thecyclic compound, either to aid in immunogenicity, or to act as a probein in vitro studies. For this purpose, any functionalizable moietycapable of reacting (e.g. making a covalent or non-covalent but strongbond) may be used. In one specific embodiment, the functionalizablemoiety is a cysteine residue which is reacted to form a disulfide bondwith an unpaired cysteine on a protein of interest, which can be, forexample, an immunogenicity enhancing agent such as Keyhole limpethemocyanin (KLH), or a carrier protein such as Bovine serum albumin(BSA) used for in vitro immunoblots or immunohistochemical assays.

The term “reacts with” as used herein generally means that there is aflow of electrons or a transfer of electrostatic charge resulting in theformation of a chemical interaction.

The term “animal” or “subject” as used herein includes all members ofthe animal kingdom including mammals, optionally including or excludinghumans.

A “conservative amino acid substitution” as used herein, is one in whichone amino acid residue is replaced with another amino acid residuewithout abolishing the protein's desired properties. Suitableconservative amino acid substitutions can be made by substituting aminoacids with similar hydrophobicity, polarity, and R-chain length for oneanother. Examples of conservative amino acid substitution include:

Conservative Substitutions Type of Amino Acid Substitutable Amino AcidsHydrophilic Ala, Pro, Gly, Glu, Asp, Gln, Asn, Ser, Thr Sulphydryl CysAliphatic Val, Ile, Leu, Met Basic Lys, Arg, His Aromatic Phe, Tyr, Trp

The term “sequence identity” as used herein refers to the percentage ofsequence identity between two polypeptide sequences or two nucleic acidsequences. To determine the percent identity of two amino acid sequencesor of two nucleic acid sequences, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in the sequence of afirst amino acid or nucleic acid sequence for optimal alignment with asecond amino acid or nucleic acid sequence). The amino acid residues ornucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % identity=number of identical overlappingpositions/total number of positions.times.100%). In one embodiment, thetwo sequences are the same length. The determination of percent identitybetween two sequences can also be accomplished using a mathematicalalgorithm. A preferred, non-limiting example of a mathematical algorithmutilized for the comparison of two sequences is the algorithm of Karlinand Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268, modifiedas in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A.90:5873-5877. Such an algorithm is incorporated into the NBLAST andXBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403. BLASTnucleotide searches can be performed with the NBLAST nucleotide programparameters set, e.g., for score=100, word length=12 to obtain nucleotidesequences homologous to a nucleic acid molecules of the presentapplication. BLAST protein searches can be performed with the XBLASTprogram parameters set, e.g., to score-50, word length=3 to obtain aminoacid sequences homologous to a protein molecule described herein. Toobtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al., 1997, Nucleic Acids Res.25:3389-3402. Alternatively, PSI-BLAST can be used to perform aniterated search which detects distant relationships between molecules(Id.). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, thedefault parameters of the respective programs (e.g., of XBLAST andNBLAST) can be used (see, e.g., the NCBI website). Another preferrednon-limiting example of a mathematical algorithm utilized for thecomparison of sequences is the algorithm of Myers and Miller, 1988,CABIOS 4:11-17. Such an algorithm is incorporated in the ALIGN program(version 2.0) which is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used. The percent identity between twosequences can be determined using techniques similar to those describedabove, with or without allowing gaps. In calculating percent identity,typically only exact matches are counted.

For antibodies, percentage sequence identities can be determined whenantibody sequences maximally aligned by IMGT or other (e.g. Kabatnumbering convention). After alignment, if a subject antibody region(e.g., the entire mature variable region of a heavy or light chain) isbeing compared with the same region of a reference antibody, thepercentage sequence identity between the subject and reference antibodyregions is the number of positions occupied by the same amino acid inboth the subject and reference antibody region divided by the totalnumber of aligned positions of the two regions, with gaps not counted,multiplied by 100 to convert to percentage.

The term “nucleic acid sequence” as used herein refers to a sequence ofnucleoside or nucleotide monomers consisting of naturally occurringbases, sugars and intersugar (backbone) linkages. The term also includesmodified or substituted sequences comprising non-naturally occurringmonomers or portions thereof. The nucleic acid sequences of the presentapplication may be deoxyribonucleic acid sequences (DNA) or ribonucleicacid sequences (RNA) and may include naturally occurring bases includingadenine, guanine, cytosine, thymidine and uracil. The sequences may alsocontain modified bases. Examples of such modified bases include aza anddeaza adenine, guanine, cytosine, thymidine and uracil; and xanthine andhypoxanthine. The nucleic acid can be either double stranded or singlestranded, and represents the sense or antisense strand. Further, theterm “nucleic acid” includes the complementary nucleic acid sequences aswell as codon optimized or synonymous codon equivalents. The term“isolated nucleic acid sequences” as used herein refers to a nucleicacid substantially free of cellular material or culture medium whenproduced by recombinant DNA techniques, or chemical precursors, or otherchemicals when chemically synthesized. An isolated nucleic acid is alsosubstantially free of sequences which naturally flank the nucleic acid(i.e. sequences located at the 5′ and 3′ ends of the nucleic acid) fromwhich the nucleic acid is derived.

“Operatively linked” is intended to mean that the nucleic acid is linkedto regulatory sequences in a manner which allows expression of thenucleic acid. Suitable regulatory sequences may be derived from avariety of sources, including bacterial, fungal, viral, mammalian, orinsect genes. Selection of appropriate regulatory sequences is dependenton the host cell chosen and may be readily accomplished by one ofordinary skill in the art. Examples of such regulatory sequencesinclude: a transcriptional promoter and enhancer or RNA polymerasebinding sequence, a ribosomal binding sequence, including a translationinitiation signal. Additionally, depending on the host cell chosen andthe vector employed, other sequences, such as an origin of replication,additional DNA restriction sites, enhancers, and sequences conferringinducibility of transcription may be incorporated into the expressionvector.

The term “vector” as used herein comprises any intermediary vehicle fora nucleic acid molecule which enables said nucleic acid molecule, forexample, to be introduced into prokaryotic and/or eukaryotic cellsand/or integrated into a genome, and include plasmids, phagemids,bacteriophages or viral vectors such as retroviral based vectors, AdenoAssociated viral vectors and the like. The term “plasmid” as used hereingenerally refers to a construct of extrachromosomal genetic material,usually a circular DNA duplex, which can replicate independently ofchromosomal DNA.

By “at least moderately stringent hybridization conditions” it is meantthat conditions are selected which promote selective hybridizationbetween two complementary nucleic acid molecules in solution.Hybridization may occur to all or a portion of a nucleic acid sequencemolecule. The hybridizing portion is typically at least 15 (e.g. 20, 25,30, 40 or 50) nucleotides in length. Those skilled in the art willrecognize that the stability of a nucleic acid duplex, or hybrids, isdetermined by the Tm, which in sodium containing buffers is a functionof the sodium ion concentration and temperature (Tm=81.5° C.−16.6 (Log10 [Na+])+0.41(% (G+C)−600/I), or similar equation). Accordingly, theparameters in the wash conditions that determine hybrid stability aresodium ion concentration and temperature. In order to identify moleculesthat are similar, but not identical, to a known nucleic acid molecule a1% mismatch may be assumed to result in about a 1° C. decrease in Tm,for example if nucleic acid molecules are sought that have a >95%identity, the final wash temperature will be reduced by about 5° C.Based on these considerations those skilled in the art will be able toreadily select appropriate hybridization conditions. In preferredembodiments, stringent hybridization conditions are selected. By way ofexample the following conditions may be employed to achieve stringenthybridization: hybridization at 5× sodium chloride/sodium citrate(SSC)/5×Denhardt's solution/1.0% SDS at Tm−5° C. based on the aboveequation, followed by a wash of 0.2×SSC/0.1% SDS at 60° C. Moderatelystringent hybridization conditions include a washing step in 3×SSC at42° C. It is understood, however, that equivalent stringencies may beachieved using alternative buffers, salts and temperatures. Additionalguidance regarding hybridization conditions may be found in: CurrentProtocols in Molecular Biology, John Wiley & Sons, N.Y., 2002, and in:Sambrook et al., Molecular Cloning: a Laboratory Manual, Cold SpringHarbor Laboratory Press, 2001.

The term “treating” or “treatment” as used herein and as is wellunderstood in the art, means an approach for obtaining beneficial ordesired results, including clinical results. Beneficial or desiredclinical results can include, but are not limited to, alleviation oramelioration of one or more symptoms or conditions, diminishment ofextent of disease, stabilized (i.e. not worsening) state of disease,preventing spread of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, diminishment of thereoccurrence of disease, and remission (whether partial or total),whether detectable or undetectable. “Treating” and “Treatment” can alsomean prolonging survival as compared to expected survival if notreceiving treatment. “Treating” and “treatment” as used herein alsoinclude prophylactic treatment. For example, a subject with early stageAD can be treated to prevent progression can be treated with a compound,antibody, immunogen, nucleic acid or composition described herein toprevent progression.

The term “administered” as used herein means administration of atherapeutically effective dose of a compound or composition of thedisclosure to a cell or subject.

As used herein, the phrase “effective amount” means an amount effective,at dosages and for periods of time necessary to achieve a desiredresult. Effective amounts when administered to a subject may varyaccording to factors such as the disease state, age, sex, weight of thesubject. Dosage regime may be adjusted to provide the optimumtherapeutic response.

The term “pharmaceutically acceptable” means that the carrier, diluent,or excipient is compatible with the other components of the formulationand not substantially deleterious to the recipient thereof.

Compositions or methods “comprising” or “including” one or more recitedelements may include other elements not specifically recited. Forexample, a composition that “comprises” or “includes” an antibody maycontain the antibody alone or in combination with other ingredients.

In understanding the scope of the present disclosure, the term“consisting” and its derivatives, as used herein, are intended to beclose ended terms that specify the presence of stated features,elements, components, groups, integers, and/or steps, and also excludethe presence of other unstated features, elements, components, groups,integers and/or steps.

The recitation of numerical ranges by endpoints herein includes allnumbers and fractions subsumed within that range (e.g. 1 to 5 includes1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood thatall numbers and fractions thereof are presumed to be modified by theterm “about.” Further, it is to be understood that “a,” “an,” and “the”include plural referents unless the content clearly dictates otherwise.The term “about” means plus or minus 0.1 to 50%, 5-50%, or 10-40%,preferably 10-20%, more preferably 10% or 15%, of the number to whichreference is being made.

Further, the definitions and embodiments described in particularsections are intended to be applicable to other embodiments hereindescribed for which they are suitable as would be understood by a personskilled in the art. For example, in the following passages, differentaspects of the invention are defined in more detail. Each aspect sodefined may be combined with any other aspect or aspects unless clearlyindicated to the contrary. In particular, any feature indicated as beingpreferred or advantageous may be combined with any other feature orfeatures indicated as being preferred or advantageous.

The singular forms of the articles “a,” “an,” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” can include a pluralityof compounds, including mixtures thereof.

III. Epitopes and Binding Proteins

The inventors have identified an epitope region in A-beta, comprisingHQK, including HHQK (SEQ ID NO: 1) at amino acids 13 to 16 on A-betapeptide and HQKL (SEQ ID NO: 20) at amino acids 14 to 17 on A-betapeptide. They have further identified that the epitope or a part thereofmay be a conformational epitope, and that HQK, HHQK (SEQ ID NO: 1)and/or HQKL (SEQ ID NO: 20) or a part thereof may be selectivelyaccessible to antibody binding in oligomeric species of A-beta.

Without wishing to be bound by theory, fibrils may present interactionsites that have a propensity to catalyze oligomerization. This may onlyoccur when selective fibril surface not present in normal individuals isexposed and able to have aberrant interactions with A-beta monomers.Environmental challenges such as low pH, osmolytes present duringinflammation, or oxidative damage may induce disruption in fibrils thatcan lead to exposure of more weakly stable regions. There is interest,then, to predict these weakly-stable regions, and use such predictionsto rationally design antibodies that could target them. Regions likelyto be disrupted in the fibril may also be good candidates for exposedregions in oligomeric species.

Computer based systems and methods to predict contiguous protein regionsthat are prone to disorder are described in U.S. Patent Application Ser.No. 62/253,044, SYSTEMS AND METHODS FOR PREDICTING MISFOLDED PROTEINEPITOPES BY COLLECTIVE COORDINATE BIASING filed Nov. 9, 2015, and U.S.patent application Ser. No. 12/574,637, “METHODS AND SYSTEMS FORPREDICTING MISFOLDED PROTEIN EPITOPES” filed Oct. 6, 2009, each of whichis hereby incorporated by reference in its entirety. As described in theExamples, the methods were applied to A-beta and identified an epitopethat as demonstrated herein is specifically and/or selectively moreaccessible in A-beta oligomers.

As described in the Examples, a cyclic peptide cyclo(CGHHQKG) (SEQ IDNO: 2) may capture the conformational differences of the epitope inoligomers relative to the monomer and/or fibril species. For example,solvent accessible surface area, curvature, conformational entropy, RMSDstructural alignment, and the dihedral angle distributions for aminoacids in the cyclic 7-mer cyclo (CGHHQKG) (SEQ ID NO: 2) were found tobe significantly different than either the fibril, or linear form of thepeptide, which may be a model of the A-beta monomer. This suggests thatthe cyclic compound may provide for a conformational epitope that isdistinct from epitope in the linear corresponding peptide, or thefibril. Antibodies raised using an immunogen comprising (CGHHQKG) (SEQID NO: 2) selectively bound cyclo(CGHHQKG) (SEQ ID NO: 2) compared tolinear CGHHQKG (SEQ ID NO: 2) and selectively bound synthetic and/ornative oligomeric A-beta species compared to monomeric A-beta and A-betafibril plaques. Further antibodies raised to cyclo(CGHHQKG) (SEQ ID NO:2) were able to inhibit in vitro propagation of A-beta aggregation. Inaddition, as demonstrated in a toxicity assay, an antibody raisedagainst (CGHHQKG) (SEQ ID NO: 2) inhibited A-beta oligomer neural celltoxicity.

II. HHQK (SEQ ID NO: 1) “Epitope” Compounds

Accordingly, the present disclosure identifies a conformational epitopein A-beta consisting of amino acids HHQK (SEQ ID NO: 1) or HQKL (SEQ IDNO: 20) or a part thereof such as HQK, HHQK (SEQ ID NO: 1) correspondingto amino acids residues 13-16 on A-beta and HQKL (SEQ ID NO: 20)corresponding to amino acids 14-17. As demonstrated in the Examples,HHQK (SEQ ID NO: 1) and HQKL (SEQ ID NO: 20) were identified as regionsprone to disorder in an A-beta fibril. The residues HHQK (SEQ ID NO:1)and HQKV (SEQ ID NO: 20) emerged in a prediction using the CollectiveCoordinates method. The residues HHQK (SEQ ID NO: 1) also emerged usingthe Promis Gō model algorithm.

An aspect includes a compound comprising an A-beta peptide comprising orconsisting of HHQK (SEQ ID NO: 1), a related epitope sequence includinga part of any of the foregoing, wherein if the peptide is HHQK (SEQ IDNO: 1), the peptide is in a conformation that is distinct in at leastone feature from linear HHQK (SEQ ID NO: 1). In an embodiment, theA-beta peptide is selected from HHQK (SEQ ID NO: 1), VHHQK (SEQ ID NO:12). HQKL (SEQ ID NO: 20) or HHQKL (SEQ ID NO: 7). The epitopes HHQKL(SEQ ID NO: 7), HQKL (SEQ ID: 20) and VHHQK (SEQ ID NO: 12), areincluded in the epitopes collectively referred to herein as HHQK (SEQ IDNO: 1) and related epitopes (and their sequences are collectivelyreferred to as related epitope sequences). In an embodiment, the relatedepitope comprises or consists of HQKL (SEQ ID NO: 20), HQK and epitopesthat comprise 1, 2 or 3 amino acids in A-beta either N-terminal and/or 1amino acid C-terminal to HQK. In an embodiment, the A-beta peptidecomprises or consists of an A-beta sequence in Table 15 (1).

In an embodiment, the compound is a cyclic compound, such as acyclopeptide.

In some embodiments, the A-beta peptide, which is optionally aconformational peptide presented for example in a cyclic compound,comprising HQK or HHQK (SEQ ID NO: 1) or a related epitope, can include1, 2 or 3 additional residues in A-beta N-terminus of and/or 1 aminoacid C-terminus of HHQK (SEQ ID NO: 1) for example HHQKL (SEQ ID NO: 7)or VHHQKL (SEQ ID NO: 6). For example, the 3 amino acids N-terminal toHHQK (SEQ ID NO: 1) in A-beta are YEV and the 3 amino acids C-terminalto HHQK (SEQ ID NO: 1) are LVF. In an embodiment, the A-beta peptide isa maximum of 6 A-beta residues. In an embodiment, the A-beta peptide isa maximum of 5 A-beta residues. In yet another embodiment A-beta peptide(e.g. in the compound such as a cyclic compound) is 4 A-beta residues,optionally HHQK (SEQ ID NO: 1).

In an embodiment, the compound further includes a linker. The linkercomprises a spacer and/or one or more functionalizable moieties. Thelinker can for example comprise 1, 2, 3, 4, 5, 6, 7 or 8 amino acidsand/or equivalently functioning molecules such as polyethylene glycol(PEG) moieties, and/or a combination thereof. In an embodiment, thespacer amino acids are selected from non-immunogenic or poorlyimmunogenic amino acid residues such as G and A, for example the spacercan be GGG, GAG, G(PEG)G, PEG-PEG(also referred to as PEG2)-GG and thelike. One or more functionalizable moieties e.g. amino acids with afunctional group may be included for example for coupling the compoundto an agent or detectable tag or a carrier such as BSA or animmunogenicity enhancing agent such as KLH.

In an embodiment the linker comprises GC-PEG, PEG-GC, GCG or PEG2-CG.

In an embodiment, the linker comprises 1, 2, 3, 4, 5, 6, 7 or 8 aminoacids.

In certain embodiments, the cyclic compound has a maximum of 12, 11, 10,9, 8, or 7 residues, optionally amino acids and/or equivalent units suchas PEG units or other similar sized chemical moieties.

In embodiments wherein the A-beta peptide comprising HQK or HHQK (SEQ IDNO: 1) includes 1, 2 or 3 additional residues found in A-beta that areN- and/or C-terminal to HHQK (SEQ ID NO: 1) the linker in the cyclizedcompound is covalently linked to the N- and/or C-termini of the A-betaresidues (e.g. where the peptide is VHHQK (SEQ ID NO: 12), the linker iscovalently linked to V and K residues). Similarly, where the A-betapeptide is HHQK (SEQ ID NO: 1), the linker is covalently linked toresidues H and K and where the A-beta peptide is HHQKL (SEQ ID NO: 7),the linker is covalently linked to residues H and L.

Proteinaceous portions of compounds (or the compound wherein the linkeris also proteinaceous) may be prepared by chemical synthesis usingtechniques well known in the chemistry of proteins such as solid phasesynthesis or synthesis in homogenous solution.

In an embodiment, the compound is a cyclic compound e.g the peptidecomprising HQK or HHQK (SEQ ID NO: 1) is comprised in a cyclic compound.

Reference to the “cyclic peptide” herein can refer to a fullyproteinaceous compound (e.g. wherein the linker is for example 1, 2, 3,4, 5, 6, 7 or 8 amino acids). It is understood that properties describedfor the cyclic peptide determined in the examples can be incorporated inother compounds (e.g. other cyclic compounds) comprising non-amino acidlinker molecules.

An aspect therefore provides a cyclic compound comprising peptide HHQK(SEQ ID NO: 1) (or a part thereof such as HQK) and a linker, wherein thelinker is covalently coupled directly or indirectly to the peptidecomprising HQK or HHQK (SEQ ID NO: 1), optionally wherein at least theone of the H, the Q and/or K residues is in an alternate conformationthan the H, Q and K residues in a linear peptide comprising HHQK (SEQ IDNO: 1), as may be manifest in A-beta monomer, and optionally wherein atleast H, Q and/or K, is in either a more constrained conformation, or analternative conformation, than the conformation occupied in a linearpeptide comprising HHQK (SEQ ID NO: 1), as may be manifest for examplein A-beta monomer.

The linear peptide comprising the A-beta sequence can be comprised in alinear compound. The linear compound or the linear peptide comprisingHHQK (SEQ ID NO: 1) is in an embodiment, a corresponding linear peptide.In another embodiment, the linear peptide is any length of A-betapeptide comprising HHQK(SEQ ID NO: 1), including for example a linearpeptide comprising A-beta residues 1-35, or smaller portions thereofsuch as A-beta residues 10-20, 11-20, 12-20, 13-20, 10-19, 10-18 and thelike etc. The linear peptide can in some embodiments also be a fulllength A-beta peptide.

In an embodiment, the cyclic compound comprises an A-beta peptidecomprising HHQK (SEQ ID NO: 1) and up to 6 A-beta residues (e.g. 1 or 2amino acids N and/or C terminus to HHQK (SEQ ID NO: 1)) and a linker,wherein the linker is covalently coupled directly or indirectly to thepeptide N-terminus residue and the C-terminus residue of the A-betapeptide and optionally wherein at least H, Q or K is in an alternateconformation than H, Q, or K in a linear peptide comprising HHQK (SEQ IDNO: 1), and/or the conformation of H, Q or K in HHQK (SEQ ID NO: 1) inthe fibril and optionally wherein at least H, Q or K, is in a moreconstrained conformation than the conformation occupied in the linearpeptide comprising HHQK (SEQ ID NO: 1).

The cyclic compound can be synthesized as a linear molecule with thelinker covalently attached to the N-terminus or C-terminus of thepeptide comprising the A-beta peptide, optionally HHQK (SEQ ID NO: 1) orrelated epitope, prior to cyclization. Alternatively part of the linkeris covalently attached to the N-terminus and part is covalently attachedto the C-terminus prior to cyclization. In either case, the linearcompound is cyclized for example in a head to tail cyclization (e.g.amide bond cyclization).

In an embodiment the cyclic compound comprises an A-beta peptidecomprising or consisting of HHQK (SEQ ID NO: 1) and a linker, whereinthe linker is coupled to the N- and C-termini of the peptide (e.g. the Hand the K residues when the peptide consists of HHQK (SEQ ID NO: 1). Inan embodiment, at least one of the H, Q and/or K residues is in analternate conformation in the cyclic compound than occupied by at leastone of the H, Q and/or K residues in a linear peptide comprising HHQK(SEQ ID NO: 1).

In an embodiment, at least one of the H, Q and/or K residues is in analternate conformation in the cyclic compound than occupied by aresidue, optionally by H, Q and/or K, in the monomer and/or fibril.

In an embodiment, at least one of the H, Q and/or K residues is in analternate conformation in the cyclic compound than occupied by a residuein the monomer and/or fibril.

In an embodiment, the alternate conformation is a constrainedconformation.

In an embodiment, at least K, optionally alone or in combination with Q,is in an alternate conformation than the conformation occupied in alinear peptide comprising HHQK (SEQ ID NO: 1) or HQKL (SEQ ID NO: 20).

For example, the alternate conformation can include one or morediffering dihedral angles in residue K16, or in residue Q15, differingfrom the dihedral angles in the linear peptide and/or peptide in thecontext of the fibril.

In an embodiment, the cyclic compound comprises a minimum averageside-chain/backbone dihedral angle difference between the cycliccompound and linear compound (e.g. linear peptide).

In an embodiment, the cyclic compound comprises a residue selected fromH, Q and K, wherein one or more side-chain or backbone dihedral anglesare at least 30 degrees, at least 40 degrees, at least 50 degrees, atleast 60 degrees, at least 70 degrees, at least 80 degrees, at least 90degrees, at least 100 degrees, at least 110 degrees, at least 120degrees, at least 130 degrees, at least 140 degrees, at least 150degrees, at least 160 degrees, at least 170 degrees, at least 180degrees, at least 190 degrees or at least 200 degrees different in thecyclic compound, than the corresponding dihedral angle in the context ofthe linear or the fibril compound.

As shown in FIG. 3, several dihedral angle distributions of Q15 and K16are substantially different in the cyclic peptide compared to the linearpeptide, or the residues in the context of the fibril 2M4J. For example,Table 3 indicates that for simulated linear peptides, cyclic peptides,and fibrils, the difference in the dihedral angle C-CA-N-HN of Q15 ismost likely about −80 degrees between cyclic and linear, and about 36degrees between cyclic and fibril. In an embodiment, the cyclic compoundcomprises a Q residue comprising an C-CA-N-HN dihedral angle that is atleast 30 degrees, at least 40 degrees, at least 50 degrees, at least 60degrees, at least 70 degrees, at least 80 degrees, than thecorresponding dihedral angle in the context of the linear peptide and/orfibril. Similarly, the differences in dihedral angles between cyclic andlinear peptides for Q15 dihedral O-C-CA-CB is most likely about 200degrees and between cyclic and fibril about 35 degrees. Accordingly inan embodiment, the cyclic compound comprises a Q comprising a dihedralangle O-C-CA-CB that is at least 30 degrees different, at least 40degrees different, at least 50 degrees different, at least 60 degreesdifferent, at least 70 degrees different, at least 80 degrees different,at least 90 degrees different, at least 100 degrees different, and so onup to at least 180 degrees different, than the corresponding dihedralangle in the context of the linear compound. The correspondingdifferences in most-likely dihedral angles between cyclic peptide andlinear peptides and cyclic peptide and fibril for K16 dihedralO-C-CA-CB, are 205 and 40 degrees respectively. Accordingly in anembodiment, the cyclic compound comprises a K comprising dihedral anglefor O-C-CA-CB that is at least 50 degrees different, at least 60 degreesdifferent, at least 70 degrees different, at least 80 degrees different,at least 90 degrees different, at least 100 degrees different, and so onup to at least 200 degrees different, than the corresponding dihedralangle in the context of either the linear peptide or the fibril.

According to the peak values of Ramachandran angles given in Table 5,the most-likely Ramachandran ϕ and ψvalues are different between thecyclic and linear peptides for residues H14, Q15, and K16. For H14, thepeak values in the cyclic distribution are (−65,−45) degrees, while thepeak values in the linear and fibril distributions are at (−145,20) and(−115,115),(−115,15) respectively. The differences Δϕ between the ϕvalues are 80, and 50 degrees, and the differences Δψ between the ψvalues are 65, 160, and 60 degrees. The ϕ,ψ values are substantiallydifferent between the linear and cyclic peptides, and fibril and cyclicpeptides. Table 5 also describes differences in ϕ,ψ angles for Q15 andK16. The difference Δϕ for Q15 between cyclic and linear 95 degrees; forΔψ for Q15 the difference between cyclic and linear is 200 degrees;between cyclic and fibril it is up to 45 degrees. For K16 the differenceΔψ is about 190 degrees between cyclic and linear; the difference Δϕ isabout 55 degrees between cyclic and fibril.

In an embodiment, the cyclic compound comprises a Q comprising anRamachandran backbone angle that is at least 30 degrees, at least 40degrees different, at least 50 degrees, at least 60 degrees, at least 70degrees, at least 80 degrees, at least 90 degrees, at least 100 degrees,at least 110 degrees, at least 120 degrees, at least 130 degrees, atleast 140 degrees, at least 150 degrees, at least 160 degrees, at least170 degrees, at least 180 degrees, at least 190 degrees, or at least 200degrees different than the corresponding Ramachandran angle in thecontext of either the linear compound and/or the fibril compound.

The angle difference can for example be positive or negative, (+) or(−).

The alternate conformation can comprise an alternate backboneorientation. For example, the backbone orientation that the cyclicepitope exposes for an antibody differs compared to linear or fibrilform.

The alternate conformation can also include an increase in or decreasein curvature centered around an amino acid or of the cyclic compoundcomprising HHQK (SEQ ID NO: 1) or a related epitope relative to a linearpeptide and/or A-beta fibril.

In an embodiment, the alternate conformation HHQK (SEQ ID NO: 1) hasaltered curvature profile relative to linear HHQK (SEQ ID NO: 1), orHHQK (SEQ ID NO: 1) in the context of the fibril structure 2M4J. Thealtered curvature profile can be seen in FIG. 2G.

The values of the curvature were determined for from N- to C-terminus H,H, Q and K in cyclo(CGHHQKG) (SEQ ID NO: 2), linear CGHHQKG (SEQ ID NO:2), and HHQK (SEQ ID NO: 1) in the context of the fibril are shown inTable 1. As described in Example 2, these were (in radians, for residuesfrom N- to C-terminus H, H, Q, and K):

Cyclic peptide: 1.49; 1.37; 0.73; 1.04

Linear Peptide: 1.46; 1.47; 1.41; 1.37 Fibril: 1.12; 1.12; 0.99; 1.15

Accordingly, the curvature in the alternate conformation, for Q, or forK, or for H, is altered by at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, or 0.7or more radians compared to the values of the curvature in the contextof the linear peptide or the fibril.

In an embodiment, Q, K, HH, HQ, QK, HHQ, HQK, and/or HHQK (SEQ ID NO: 1)are in an alternate conformation, for example as compared to what isoccupied by these residues in a non-oligomeric conformation, such as thelinear peptide and/or fibril.

FIG. 2A plots the curvature for linear CGHHQKG (SEQ ID NO: 2) asobtained from different equilibrium simulation times. The legend showsseveral curves that start from 10 ns and continue to 72 ns, 134 ns, 196ns, or 258 ns. As simulation time is increased, the curvature valuesconverge to the values reported above and in Table 1. Similar studiesare shown in FIG. 2B for the cyclic peptide and FIG. 2C for the fibril.Panels D, E, and F show the convergence in the sum of the curvaturevalues as a function of simulation time, for the linear, cyclic, andfibril conformations respectively. The degree of convergence indicatesthat the error bars are approximately 0.007 radian for the cyclicpeptide, 0.011 radian for the linear peptide, and 0.005 radian for thefibril.

Cyclic compounds which show similar changes are also encompassed.

The cyclic compound in some embodiments that comprises an A-beta peptidecomprising HHQK (SEQ ID NO: 1), HQK or HQKL (SEQ ID NO: 20) can include1, or 2 or more residues in A-beta upstream and/or downstream of one ofthe foregoing, for example of HHQK (SEQ ID NO: 1). In such cases thespacer is covalently linked to the N- and C-termini of the ends of thecorresponding residues of the A-beta sequence.

In some embodiments, the linker or spacer is indirectly coupled to theN- and C-terminus residues of the A-beta peptide.

In an embodiment, the cyclic compound is a compound in FIG. 7C.

Methods for making cyclized peptides are known in the art and includeSS-cyclization or amide cyclization (head-to-tail, or backbonecyclization). Methods are further described in Example 3. For example, apeptide with “C” residues at its N- and C-termini, e.g. CGHHQKGC (SEQ IDNO: 13), can be reacted by SS-cyclization to produce a cyclic peptide.As described in Example 2, a cyclic compound of FIG. 7C was assessed forits relatedness to the conformational epitope identified. The cycliccompound comprising HHQK (SEQ ID NO: 1) peptide for example can be usedto raise antibodies selective for one or more conformational features.

The epitope HHQK (SEQ ID NO: 1) and/or a part thereof, as describedherein may be a potential target in misfolded propagating strains ofA-beta involved in A-beta spreading pathology, and antibodies thatrecognize the conformational epitope may for example be useful indetecting such propagating strains.

Also provided in another aspect is an isolated peptide comprising anA-beta peptide sequence described herein, including linear peptides andcyclic peptides. Linear peptides can for example be used for selectingantibodies that lack specific or selective binding thereto. The isolatedpeptide can comprise a linker sequence described herein. The linker canbe covalently coupled to the N or C terminus or may be partially coupledto the N terminus and partially coupled to the C terminus as in CGHHQKG(SEQ ID NO: 2) linear peptide. In the cyclic peptide, the linker iscoupled to the C-terminus and N-terminus directly or indirectly.

Another aspect includes an immunogen comprising a compound, optionally acyclic compound described herein. The immunogen may comprise for exampleHQK or HHQK (SEQ ID NO: 1) or a related epitope sequence presented in acyclic compound. The A-beta peptide may comprise additional A-betasequence. The amino acids may be directly upstream and/or downstreamsaid sequences. Antibodies raised against such immunogens can beselected for example for binding to a cyclopeptide comprising HHQK (SEQID NO: 1) or a related epitope.

In an embodiment, the immunogen is a cyclic peptide comprising A-betapeptide HHQK or a related epitope sequence.

In an embodiment, the immunogen comprises immunogenicity enhancing agentsuch as Keyhole Limpet Hemocyanin (KLH). The immunogenicity enhancingagent can be coupled to the compound either directly, such as through anamide bound, or indirectly through a chemical linker.

The immunogen can be produced by conjugating the cyclic compoundcontaining the constrained epitope peptide to an immunogenicityenhancing agent such as Keyhole Limpet Hemocyanin (KLH) or a carriersuch bovine serum albumin (BSA) using for example the method describedin Lateef et al 2007, herein incorporated by reference. In anembodiment, the method described in Example 3 is used.

An immunogen is suitably prepared or formulated for administration to asubject, for example, the immunogen may be sterile, or purified.

A further aspect is an isolated nucleic acid encoding the proteinaceousportion of a compound or immunogen described herein.

In embodiment, the nucleic acid molecule encodes any one of the aminoacid sequences sent forth herein, optionally in SEQ ID NOS: 1-21 orTable 15 (1).

In an embodiment, nucleic acid molecule encodes HHQK (SEQ ID NO: 1) or arelated epitope and optionally a linker described herein.

A further aspect is a vector comprising said nucleic acid. Suitablevectors are described elsewhere herein.

III. Antibodies Cells and Nucleic Acids

As demonstrated in Examples 6 and 7, the cyclic compound CGHHQKG (SEQ IDNO: 2) was immunogenic, and produced a number of antibodies thatselectively bind the cyclic compound relative to the correspondinglinear peptide. As described herein, antibodies raised usingcyclo(CGHHQKG) (SEQ ID NO: 2) included antibodies that were selectivefor the cyclic compound, selectively bound A-beta oligomer over monomer,and lacked appreciable plaque staining in AD tissue. The epitope HHQK(SEQ ID NO: 1) and/or a part thereof, as described herein may be apotential target in misfolded propagating strains of A-beta involved inAD, and antibodies that recognize the conformational epitope may forexample be useful in detecting such propagating strains. Furtherantibodies raised to the cyclic compound inhibited A-beta aggregationand also inhibited A-beta oligomer induced neural cell toxicitysuggesting their use as therapeutics.

Accordingly, the compounds and particularly the cyclic compoundsdescribed above can be used to raise antibodies that specifically bindHQ, HQK, QK, and/or HHQK (SEQ ID NO: 1) in A-beta and/or which recognizespecific conformations of these residues in A-beta, including one ormore differential features described herein. Similarly cyclic compoundscomprising for example HKLV (SEQ ID NO: 20), VHHQK (SEQ ID NO: 12),HHQKL (SEQ ID NO: 7), VHHQKL (SED ID NO: 6) and/or other related epitopesequences described herein can be used to raise antibodies thatspecifically bind HQK, HHQK (SEQ ID NO: 1), HQKL (SEQ ID NO: 20) etcand/or specific conformational epitopes thereof.

Accordingly, an aspect includes an antibody (including a bindingfragment thereof) that specifically binds to an A-beta peptide having asequence HQK, HHQK (SEQ ID NO: 1) or a related epitope sequencedescribed herein, optionally an A-beta sequence in Table 15 (1).

In an embodiment, the A-beta peptide is comprised in a cyclic compound,optionally a cyclic peptide and the antibody is specific and/orselective for A-beta presented in the cyclic compound.

In an embodiment, the cyclic compound is a cyclic peptide optionally onedescribed herein, such as set forth in SEQ ID NO: 2, 3 or 4. The termscyclopeptide and cyclic peptide are used interchangeably herein.

In an embodiment, the antibody specifically and/or selectively binds theA-beta peptide presented in the cyclic compound relative to acorresponding linear compound. In an embodiment, the antibody isselective for the A-beta peptide as presented in the cyclic compoundrelative to a corresponding linear compound comprising the A-betapeptide.

In an embodiment, the antibody does not bind a linear peptide comprisingthe sequence HHQK (SEQ ID NO: 1), optionally wherein the sequence of thelinear peptide is a linear version of a cyclic sequence used to raisethe antibody, optionally as set forth in SEQ ID NOs: 2, 3, 4 or 32.

In an embodiment, the antibody specifically binds an epitope on A-beta,the epitope comprising or consisting HHQK (SEQ ID NO: 1), a relatedepitope thereof or a part thereof or a conformational epitope of any ofthe foregoing. In an embodiment, wherein when the epitope consists ofHHQK (SEQ ID NO: 1) it is a conformational epitope.

As described in the examples, antibodies having one or properties can beselected using assays described in the Examples.

In an embodiment the antibody is isolated. In an embodiment, theantibody is an exogenous antibody.

In an embodiment, the antibody does not specifically bind and/or is notselective for linear HQKLVF (SEQ ID NO: 14), linear HQKLVFF (SEQ ID NO:15), linear HQKLVFFAED (SEQ ID NO: 16), linear EVHHQK (SEQ ID NO: 18),linear VHHQK (SEQ ID NO: 12), or linear HHQKLVFFAEDVGSNK (SEQ ID NO: 19)relative to cyclic compound comprising an A-beta peptide consisting ofHHQK (SEQ ID NO:1), HQK or HQKL (SEQ ID NO: 20). In an embodiment, theantibody does not specifically bind and/or is not selective for linearpeptides consisting of HHQK (SEQ ID NO: 1). Selective binding can bemeasured using an ELISA or surface plasmon resonance measurement, asdescribed herein.

III. Antibodies, Cells and Nucleic Acids

As demonstrated in the examples, antibodies raised using an immunogencomprising (CGHHQKG) (SEQ ID NO: 2) selectively bound cyclo(CGHHQKG)(SEQ ID NO: 2) compared to linear CGHHQKG (SEQ ID NO: 2) and selectivelybound synthetic and/or native oligomeric A-beta species compared tomonomeric A-beta and A-beta fibril plaques. Further antibodies raised tocyclo(CGHHQKG) (SEQ ID NO: 2) were able to inhibit in vitro propagationof A-beta aggregation. In addition, as demonstrated in a toxicity assay,an antibody raised against (CGHHQKG) (SEQ ID NO: 2) inhibited A-betaoligomer neural cell toxicity.

Accordingly a further aspect is an antibody which specifically binds anepitope present on A-beta, wherein the epitope comprises or consists ofat least one amino acid residue predominantly involved in binding to theantibody, wherein the at least one amino acid is H, Q, or K embeddedwithin the sequence HHQK (SEQ ID NO:1), HQK or HQKL (SEQ ID NO: 20),optionally wherein the epitope when consisting of HHQK (SEQ ID NO:1) isa conformational epitope (e.g. selectively binds an A-beta peptide in analternate optionally constrained conformation relative to thecorresponding linear peptide, for example where at least one amino acidof the epitope is more constrained). In an embodiment, the epitopecomprises or consists of at least two consecutive amino acid residuespredominantly involved in binding to the antibody, wherein the at leasttwo consecutive amino acids are HQ, or QK embedded within HHQK (SEQ IDNO:1) HQK or HQKL (SEQ ID NO: 20).

In another embodiment, the epitope recognized is a conformationalepitope and consists of HHQK (SEQ ID NO: 1), HQK or HQKL (SEQ ID NO:20). In an embodiment, the antibody selectively binds HHQK (SEQ IDNO: 1) in a cyclic peptide, optionally cyclo(CGHHQKG) (SEQ ID NO: 2)relative to a corresponding linear peptide.

In an embodiment, the antibody is a conformation selective antibody. Inan embodiment, the antibody specifically and/or selectively binds acyclic compound comprising an epitope peptide sequence described hereincompared to the corresponding linear sequence. For example an antibodythat binds a particular epitope conformation can be referred to as aconformation specific antibody. Such antibodies can be selected usingthe methods described herein. The conformation selective antibody candifferentially recognize a particular A-beta species or a group ofrelated species (e.g. dimers, trimers, and other oligomeric species) andcan have a higher affinity for one species or group of species comparedto another (e.g. to either the monomer or fibril species).

In an embodiment, the antibody does not specifically bind monomericA-beta. In an embodiment, the antibody does not specifically bind A-betasenile plaques, for example in situ in AD brain tissue.

In another embodiment, the antibody does not selectively bind monomericA-beta compared to native- or synthetic-oligomeric A-beta.

In an embodiment, the antibody specifically binds a cyclic compoundcomprising an epitope peptide sequence described herein comprising atleast one alternate conformational feature described herein (e.g. of theepitope in a cyclic compound compared to a linear compound).

For example, in an embodiment, the antibody specifically binds a cycliccompound comprises a residue selected from H, Q and K, wherein at leastone dihedral angle is at least 30 degrees, at least 40 degrees, at least50 degrees, at least 60 degrees, at least 70 degrees, at least 80degrees, at least 90 degrees, at least 100 degrees, at least 110degrees, at least 120 degrees, at least 130 degrees, at least 140degrees at least 150 degrees different in the cyclic compound, than thecorresponding dihedral angle in the context of the linear compound.

In an embodiment, the antibody selectively binds a cyclic compoundcomprising HHQK (SEQ ID NO: 1) or a part thereof, optionally in thecontext of cyclo(CGHHQKG) (SEQ ID NO: 2) relative to a linear peptidecomprising HHQK (SEQ ID NO: 1), optionally in the context of linearCGHHQKG (SEQ ID NO: 2). For example, in an embodiment the antibodyselectively binds HHQK (SEQ ID NO: 1) or related epitope sequence in acyclic conformation and has at least 2 fold, at least 5 fold, at least10 fold at least 20 fold, at least 30 fold, at least 40 fold, at least50 fold, at least 100 fold, at least 500 fold, at least 1000 fold moreselective for HHQK (SEQ ID NO: 1) in the cyclic conformation compared toHHQK (SEQ ID NO: 1) in a linear compound such as a corresponding linearcompound, for example as measured by ELISA or surface plasmon resonance,optionally using a method described herein.

In an embodiment, the antibody selectively binds a cyclic compoundcomprising the epitope sequence relative to linear peptide or a speciesof A-beta such as A-beta oligomer relative to monomer. In an embodiment,the selectivity is at least 2 fold, at least 3 fold, at least 5 fold, atleast 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, atleast 50 fold, at least 100 fold, at least 500 fold, at least 1000 foldmore selective for the cyclic compound and/or A-beta oligomer over aspecies of A-beta selected from A-beta monomer and/or A-beta fibriland/or linear HHQK (SEQ ID NO: 1), optionally linear CGHHQKG (SEQ ID NO:2).

In an embodiment, the A-beta oligomer comprises A-beta 1-42 subunits.

In an embodiment, the antibody lacks A-beta fibril plaque (also referredto as senile plaque) staining. Absence of plaque staining can beassessed by comparing to a positive control such as A-beta-specificantibodies 6E10 and 4G8 (Biolegend, San Diego, Calif.), or 2C8 (EnzoLife Sciences Inc., Farmingdale, N.Y.) and an isotype control. Anantibody described herein lacks or has negligible A-beta fibril plaquestaining if the antibody does not show typical plaque morphologystaining and the level of staining is comparable to or no more than 2fold the level seen with an IgG negative isotype control. The scale canfor example set the level of staining with isotype control at 1 and with6E10 at 10. An antibody lacks A-beta fibril plaque staining if the levelof staining on such a scale is 2 or less. In embodiment, the antibodyshows minimal A-beta fibril plaque staining, for example on theforegoing scale, levels scored at less about or less than 3.

In an embodiment, the antibody is produced using a cyclic compound orimmunogen described herein, optionally using a method described herein.

In an embodiment, the antibody is a monoclonal antibody.

To produce monoclonal antibodies, antibody producing cells (lymphocytes)can be harvested from a subject immunized with an immunogen describedherein, and fused with myeloma cells by standard somatic cell fusionprocedures thus immortalizing these cells and yielding hybridoma cells.Such techniques are well known in the art, (e.g. the hybridoma techniqueoriginally developed by Kohler and Milstein (Nature 256:495-497 (1975))as well as other techniques such as the human B-cell hybridoma technique(Kozbor et al., Immunol. Today 4:72 (1983)), the EBV-hybridoma techniqueto produce human monoclonal antibodies (Cole et al., Methods Enzymol,121: 140-67 (1986)), and screening of combinatorial antibody libraries(Huse et al., Science 246:1275 (1989)). Hybridoma cells can be screenedimmunochemically for production of antibodies specifically reactive withthe desired epitopes and the monoclonal antibodies can be isolated.

Specific antibodies, or antibody fragments, reactive against particularantigens or molecules, may also be generated by screening expressionlibraries encoding immunoglobulin genes, or portions thereof, expressedin bacteria with cell surface components. For example, complete Fabfragments, VH regions and FV regions can be expressed in bacteria usingphage expression libraries (see for example Ward et al., Nature41:544-546 (1989); Huse et al., Science 246:1275-1281 (1989); andMcCafferty et al., Nature 348:552-554 (1990).

In an embodiment, the antibody is a humanized antibody.

The humanization of antibodies from non-human species has been welldescribed in the literature. See for example EP-B1 0 239400 and Carter &Merchant 1997 (Curr Opin Biotechnol 8, 449-454, 1997 incorporated byreference in their entirety herein). Humanized antibodies are alsoreadily obtained commercially (eg. Scotgen Limited, 2 Holly Road,Twickenham, Middlesex, Great Britain.).

Humanized forms of rodent antibodies are readily generated by CDRgrafting (Riechmann et al. Nature, 332:323-327, 1988). In this approachthe six CDR loops comprising the antigen binding site of the rodentmonoclonal antibody are linked to corresponding human framework regions.CDR grafting often yields antibodies with reduced affinity as the aminoacids of the framework regions may influence antigen recognition (Foote& Winter. J Mol Biol, 224: 487-499, 1992). To maintain the affinity ofthe antibody, it is often necessary to replace certain frameworkresidues by site directed mutagenesis or other recombinant techniquesand may be aided by computer modeling of the antigen binding site (Co etal. J Immunol, 152: 2968-2976, 1994).

Humanized forms of antibodies are optionally obtained by resurfacing(Pedersen et al. J Mol Biol, 235: 959-973, 1994). In this approach onlythe surface residues of a rodent antibody are humanized.

Human antibodies specific to a particular antigen may be identified by aphage display strategy (Jespers et al. Bio/Technology, 12: 899-903,1994). In one approach, the heavy chain of a rodent antibody directedagainst a specific antigen is cloned and paired with a repertoire ofhuman light chains for display as Fab fragments on filamentous phage.The phage is selected by binding to antigen. The selected human lightchain is subsequently paired with a repertoire of human heavy chains fordisplay on phage, and the phage is again selected by binding to antigen.The result is a human antibody Fab fragment specific to a particularantigen. In another approach, libraries of phage are produced wheremembers display different human antibody fragments (Fab or Fv) on theirouter surfaces (Dower et al., WO 91/17271 and McCafferty et al., WO92/01047). Phage displaying antibodies with a desired specificity areselected by affinity enrichment to a specific antigen. The human Fab orFv fragment identified from either approach may be recloned forexpression as a human antibody in mammalian cells.

Human antibodies are optionally obtained from transgenic animals (U.S.Pat. Nos. 6,150,584; 6,114,598; and 5,770,429). In this approach theheavy chain joining region (JH) gene in a chimeric or germ-line mutantmouse is deleted. Human germ-line immunoglobulin gene array issubsequently transferred to such mutant mice. The resulting transgenicmouse is then capable of generating a full repertoire of humanantibodies upon antigen challenge.

Humanized antibodies are typically produced as antigen binding fragmentssuch as Fab, Fab′ F(ab′)2, Fd, Fv and single domain antibody fragments,or as single chain antibodies in which the heavy and light chains arelinked by a spacer. Also, the human or humanized antibodies may exist inmonomeric or polymeric form. The humanized antibody optionally comprisesone non-human chain and one humanized chain (i.e. one humanized heavy orlight chain).

Antibodies including humanized or human antibodies are selected from anyclass of immunoglobulins including: IgM, IgG, IgD, IgA or IgE; and anyisotype, including: IgG1, IgG2, IgG3 and IgG4. The humanized or humanantibody may include sequences from one or more than one isotype orclass.

Additionally, antibodies specific for the epitopes described herein arereadily isolated by screening antibody phage display libraries. Forexample, an antibody phage library is optionally screened by using adisease specific epitope of the current invention to identify antibodyfragments specific for the disease specific epitope. Antibody fragmentsidentified are optionally used to produce a variety of recombinantantibodies that are useful with different embodiments of the presentinvention. Antibody phage display libraries are commercially available,for example, through Xoma (Berkeley, Calif.) Methods for screeningantibody phage libraries are well known in the art.

A further aspect is antibody and/or binding fragment thereof comprisinga light chain variable region and a heavy chain variable region, theheavy chain variable region comprising complementarity determiningregions CDR-H1, CDR-H2 and CDR-H3, the light chain variable regioncomprising complementarity determining region CDR-L1, CDR-L2 and CDR-L3and with the amino acid sequences of said CDRs comprising the sequencesset forth below.

CDR-H1 (SEQ ID NO: 22) GYSFTSYW CDR-H2 (SEQ ID NO: 23) VHPGRGVST CDR-H3(SEQ ID NO: 24) SRSHGNTYWFFDV CDR-L1 (SEQ ID NO: 25) QSIVHSNGNTY CDR-L2(SEQ ID NO: 26) KVS CDR-L3 (SEQ ID NO: 27) FQGSHVPFT

In an embodiment, the antibody is a monoclonal antibody. In anembodiment, the antibody is a chimeric antibody such as a humanizedantibody comprising the CDR sequences as recited in Table 13.

Also provided in another embodiment, is an antibody comprising the CDRsin Table 13 and a light chain variable region and a heavy chain variableregion, optionally in the context of a single chain antibody.

In yet another aspect, the antibody comprises a heavy chain variableregion comprises: i) an amino acid sequence as set forth in SEQ ID NO:29; ii) an amino acid sequence with at least 50%, at least 60%, at least70%, at least 80% or at least 90% sequence identity to SEQ ID NO: 29,wherein the CDR sequences are as set forth in SEQ ID NO: 22, 23 and 24,or iii) a conservatively substituted amino acid sequence i). In anotheraspect the antibody comprises a light chain variable region comprisingi) an amino acid sequence as set forth in SEQ ID NO: 31, ii) an aminoacid sequence with at least 50%, at least 60%, at least 70%, at least80% or at least 90% sequence identity to SEQ ID NO: 31, wherein the CDRsequences are as set forth in SEQ ID NO: 25, 26 and 27, or iii) aconservatively substituted amino acid sequence of i). In anotherembodiment, the heavy chain variable region amino acid sequence isencoded by a nucleotide sequence as set out in SEQ ID NO: 28 or a codondegenerate optimized version thereof. In another embodiment, theantibody comprises a light chain variable region amino acid sequenceencoded by a nucleotide sequence as set out in SEQ ID NO: 30 or a codondegenerate or optimized version thereof. In an embodiment, the heavychain variable region comprises an amino acid sequence as set forth inSEQ ID NO: 29. In an embodiment, the light chain variable regioncomprises an amino acid sequence as set forth in SEQ ID NO: 31.

Another aspect is an antibody that specifically binds a same epitope asthe antibody with CDR sequences as recited in Table 13.

Another aspect includes an antibody that competes for binding to humanA-beta with an antibody comprising the CDR sequences as recited in Table13.

Competition between antibodies can be determined for example using anassay in which an antibody under test is assessed for its ability toinhibit specific binding of a reference antibody to the common antigen.A test antibody competes with a reference antibody if an excess of atest antibody (e.g., at least a 2 fold, 5, fold, 10 fold or 20 fold)inhibits binding of the reference antibody by at least 50%, at least75%, at least 80%, at least 90% or at least 95% as measured in acompetitive binding assay.

A further aspect is an antibody conjugated to a therapeutic, detectablelabel or cytotoxic agent. In an embodiment, the detectable label is apositron-emitting radionuclide. A positron-emitting radionuclide can beused for example in PET imaging.

A further aspect relates to an antibody complex comprising an antibodydescribed herein and/or a binding fragment thereof and oligomericA-beta.

A further aspect is an isolated nucleic acid encoding an antibody orpart thereof described herein.

Nucleic acids encoding a heavy chain or a light chain are also provided,for example encoding a heavy chain comprising CDR-H1, CDR-H2 and/orCDR-H3 regions described herein or encoding a light chain comprisingCDR-L1, CDR-L2 and/or CDR-L3 regions described herein.

The present disclosure also provides variants of the nucleic acidsequences that encode for the antibody and/or binding fragment thereofdisclosed herein. For example, the variants include nucleotide sequencesthat hybridize to the nucleic acid sequences encoding the antibodyand/or binding fragment thereof disclosed herein under at leastmoderately stringent hybridization conditions or codon degenerate oroptimized sequences In another embodiment, the variant nucleic acidsequences have at least 50%, at least 60%, at least 70%, most preferablyat least 80%, even more preferably at least 90% and even most preferablyat least 95% sequence identity to nucleic acid sequences encoding SEQ IDNOs: 29 and 31.

A further aspect is an isolated nucleic acid encoding an antibodydescribed herein.

Another aspect is an expression cassette or vector comprising thenucleic acid herein disclosed. In an embodiment, the vector is anisolated vector.

The vector can be any vector, including vectors suitable for producingan antibody and/or binding fragment thereof or expressing a peptidesequence described herein.

The nucleic acid molecules may be incorporated in a known manner into anappropriate expression vector which ensures expression of the protein.Possible expression vectors include but are not limited to cosmids,plasmids, or modified viruses (e.g. replication defective retroviruses,adenoviruses and adeno-associated viruses). The vector should becompatible with the host cell used. The expression vectors are “suitablefor transformation of a host cell”, which means that the expressionvectors contain a nucleic acid molecule encoding the peptidescorresponding to epitopes or antibodies described herein.

In an embodiment, the vector is suitable for expressing for examplesingle chain antibodies by gene therapy. The vector can be adapted forspecific expression in neural tissue, for example using neural specificpromoters and the like. In an embodiment, the vector comprises an IRESand allows for expression of a light chain variable region and a heavychain variable region. Such vectors can be used to deliver antibody invivo.

Suitable regulatory sequences may be derived from a variety of sources,including bacterial, fungal, viral, mammalian, or insect genes.

Examples of such regulatory sequences include: a transcriptionalpromoter and enhancer or RNA polymerase binding sequence, a ribosomalbinding sequence, including a translation initiation signal.Additionally, depending on the host cell chosen and the vector employed,other sequences, such as an origin of replication, additional DNArestriction sites, enhancers, and sequences conferring inducibility oftranscription may be incorporated into the expression vector.

In an embodiment, the regulatory sequences direct or increase expressionin neural tissue and/or cells.

In an embodiment, the vector is a viral vector.

The recombinant expression vectors may also contain a marker gene whichfacilitates the selection of host cells transformed, infected ortransfected with a vector for expressing an antibody or epitope peptidedescribed herein.

The recombinant expression vectors may also contain expression cassetteswhich encode a fusion moiety (i.e. a “fusion protein”) which providesincreased expression or stability of the recombinant peptide; increasedsolubility of the recombinant peptide; and aid in the purification ofthe target recombinant peptide by acting as a ligand in affinitypurification, including for example tags and labels described herein.Further, a proteolytic cleavage site may be added to the targetrecombinant protein to allow separation of the recombinant protein fromthe fusion moiety subsequent to purification of the fusion protein.Typical fusion expression vectors include pGEX (Amrad Corp., Melbourne,Australia), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5(Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase(GST), maltose E binding protein, or protein A, respectively, to therecombinant protein.

Systems for the transfer of genes for example into neurons and neuraltissue both in vitro and in vivo include vectors based on viruses, mostnotably Herpes Simplex Virus, Adenovirus, Adeno-associated virus (AAV)and retroviruses including lentiviruses. Alternative approaches for genedelivery include the use of naked, plasmid DNA as well as liposome-DNAcomplexes. Another approach is the use of AAV plasmids in which the DNAis polycation-condensed and lipid entrapped and introduced into thebrain by intracerebral gene delivery (Leone et al. US Application No.2002076394).

Accordingly, in another aspect, the compounds, immunogens, nucleicacids, vectors and antibodies described herein may be formulated invesicles such as liposomes, nanoparticles, and viral protein particles,for example for delivery of antibodies, compounds, immunogens andnucleic acids described herein. In particular synthetic polymervesicles, including polymersomes, can be used to administer antibodies.

Also provided in another aspect is a cell, optionally an isolated and/orrecombinant cell, expressing an antibody described herein or comprisinga vector herein disclosed.

The recombinant cell can be generated using any cell suitable forproducing a polypeptide, for example suitable for producing an antibodyand/or binding fragment thereof. For example to introduce a nucleic acid(e.g. a vector) into a cell, the cell may be transfected, transformed orinfected, depending upon the vector employed.

Suitable host cells include a wide variety of prokaryotic and eukaryotichost cells. For example, the proteins described herein may be expressedin bacterial cells such as E. coli, insect cells (using baculovirus),yeast cells or mammalian cells.

In an embodiment, the cell is a eukaryotic cell selected from a yeast,plant, worm, insect, avian, fish, reptile and mammalian cell.

In another embodiment, the mammalian cell is a myeloma cell, a spleencell, or a hybridoma cell.

In an embodiment, the cell is a neural cell.

Yeast and fungi host cells suitable for expressing an antibody orpeptide include, but are not limited to Saccharomyces cerevisiae,Schizosaccharomyces pombe, the genera Pichia or Kluyveromyces andvarious species of the genus Aspergillus. Examples of vectors forexpression in yeast S. cerivisiae include pYepSec1, pMFa, pJRY88, andpYES2 (Invitrogen Corporation, San Diego, Calif.). Protocols for thetransformation of yeast and fungi are well known to those of ordinaryskill in the art.

Mammalian cells that may be suitable include, among others: COS (e.g.,ATCC No. CRL 1650 or 1651), BHK (e.g. ATCC No. CRL 6281), CHO (ATCC No.CCL 61), HeLa (e.g., ATCC No. CCL 2), 293 (ATCC No. 1573) and NS-1cells. Suitable expression vectors for directing expression in mammaliancells generally include a promoter (e.g., derived from viral materialsuch as polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40), aswell as other transcriptional and translational control sequences.Examples of mammalian expression vectors include pCDM8 and pMT2PC.

In an embodiment, the cell is a fused cell such as a hybridoma cell, thehybridoma cell producing an antibody specific and/or selective for anepitope or epitope sequence described herein, including for example thatselectively binds A-beta oligomers over A-beta monomers, selectivelybinds an epitope sequence presented in a cyclic compound relative to alinear compound or lacks or has negligible plaque binding.

A further aspect is a hybridoma cell line producing an antibody specificfor an epitope described herein.

IV. Compositions

A further aspect is a composition comprising a compound, immunogen,nucleic acid, vector or antibody described herein.

In an embodiment, the composition comprises a diluent.

Suitable diluents for nucleic acids include but are not limited towater, saline solutions and ethanol.

Suitable diluents for polypeptides, including antibodies or fragmentsthereof and/or cells include but are not limited to saline solutions, pHbuffered solutions and glycerol solutions or other solutions suitablefor freezing polypeptides and/or cells.

In an embodiment, the composition is a pharmaceutical compositioncomprising any of the peptides, immunogens, antibodies, nucleic acids orvectors disclosed herein, and optionally comprising a pharmaceuticallyacceptable carrier.

The compositions described herein can be prepared by per se knownmethods for the preparation of pharmaceutically acceptable compositionsthat can be administered to subjects, optionally as a vaccine, such thatan effective quantity of the active substance is combined in a mixturewith a pharmaceutically acceptable vehicle.

Pharmaceutical compositions include, without limitation, lyophilizedpowders or aqueous or non-aqueous sterile injectable solutions orsuspensions, which may further contain antioxidants, buffers,bacteriostats and solutes that render the compositions substantiallycompatible with the tissues or the blood of an intended recipient. Othercomponents that may be present in such compositions include water,surfactants (such as Tween), alcohols, polyols, glycerin and vegetableoils, for example. Extemporaneous injection solutions and suspensionsmay be prepared from sterile powders, granules, tablets, or concentratedsolutions or suspensions. The composition may be supplied, for examplebut not by way of limitation, as a lyophilized powder which isreconstituted with sterile water or saline prior to administration tothe patient.

Pharmaceutical compositions may comprise a pharmaceutically acceptablecarrier. Suitable pharmaceutically acceptable carriers includeessentially chemically inert and nontoxic compositions that do notinterfere with the effectiveness of the biological activity of thepharmaceutical composition. Examples of suitable pharmaceutical carriersinclude, but are not limited to, water, saline solutions, glycerolsolutions, ethanol, N-(1(2,3-dioleyloxy)propyl)N,N,N-trimethylammoniumchloride (DOTMA), diolesylphosphotidyl-ethanolamine (DOPE), andliposomes. Such compositions should contain a therapeutically effectiveamount of the compound, together with a suitable amount of carrier so asto provide the form for direct administration to the patient.

The composition may be in the form of a pharmaceutically acceptable saltwhich includes, without limitation, those formed with free amino groupssuch as those derived from hydrochloric, phosphoric, acetic, oxalic,tartaric acids, etc., and those formed with free carboxyl groups such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylarnino ethanol,histidine, procaine, etc.

In an embodiment comprising a compound or immunogen described herein,the composition comprises an adjuvant.

Adjuvants that can be used for example, include Intrinsic adjuvants(such as lipopolysaccharides) normally are the components of killed orattenuated bacteria used as vaccines. Extrinsic adjuvants areimmunomodulators which are typically non-covalently linked to antigensand are formulated to enhance the host immune responses. Aluminumhydroxide, aluminum sulfate and aluminum phosphate (collectivelycommonly referred to as alum) are routinely used as adjuvants. A widerange of extrinsic adjuvants can provoke potent immune responses toimmunogens. These include saponins such as Stimulons (QS21, Aquila,Worcester, Mass.) or particles generated therefrom such as ISCOMs and(immunostimulating complexes) and ISCOMATRIX, complexed to membraneprotein antigens (immune stimulating complexes), pluronic polymers withmineral oil, killed mycobacteria and mineral oil, Freund's completeadjuvant, bacterial products such as muramyl dipeptide (MDP) andlipopolysaccharide (LPS), as well as lipid A, and liposomes.

In an embodiment, the adjuvant is aluminum hydroxide. In anotherembodiment, the adjuvant is aluminum phosphate. Oil in water emulsionsinclude squalene; peanut oil; MF59 (WO 90/14387); SAF (SyntexLaboratories, Palo Alto, Calif.); and Ribi™ (Ribi Immunochem, Hamilton,Mont.). Oil in water emulsions may be used with immunostimulating agentssuch as muramyl peptides (for example,N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP),-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutamyl-L-alanine-2-(1′-2′dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(MTP-PE),N-acetylglucsaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxypropylamide (DTP-DPP) theramide (TM)), or other bacterial cell wallcomponents.

The adjuvant may be administered with an immunogen as a singlecomposition. Alternatively, an adjuvant may be administered before,concurrent and/or after administration of the immunogen.

Commonly, adjuvants are used as a 0.05 to 1.0 percent solution inphosphate—buffered saline. Adjuvants enhance the immunogenicity of animmunogen but are not necessarily immunogenic themselves. Adjuvants mayact by retaining the immunogen locally near the site of administrationto produce a depot effect facilitating a slow, sustained release ofimmunogen to cells of the immune system. Adjuvants can also attractcells of the immune system to an immunogen depot and stimulate suchcells to elicit immune responses. As such, embodiments may encompasscompositions further comprising adjuvants.

Adjuvants for parenteral immunization include aluminum compounds (suchas aluminum hydroxide, aluminum phosphate, and aluminum hydroxyphosphate). The antigen can be precipitated with, or adsorbed onto, thealuminum compound according to standard protocols. Other adjuvants suchas RIBI (ImmunoChem, Hamilton, Mont.) can also be used in parenteraladministration.

Adjuvants for mucosal immunization include bacterial toxins (e.g., thecholera toxin (CT), the E. coli heat-labile toxin (LT), the Clostridiumdifficile toxin A and the pertussis toxin (PT), or combinations,subunits, toxoids, or mutants thereof). For example, a purifiedpreparation of native cholera toxin subunit B (CTB) can be of use.Fragments, homologs, derivatives, and fusion to any of these toxins arealso suitable, provided that they retain adjuvant activity. Preferably,a mutant having reduced toxicity is used. Suitable mutants have beendescribed (e.g., in WO 95/17211 (Arg-7-Lys CT mutant), WO 96/6627(Arg-192-Gly LT mutant), and WO 95/34323 (Arg-9-Lys and Glu-129-Gly PTmutant)). Additional LT mutants that can be used in the methods andcompositions include, for example Ser-63-Lys, Ala-69-Gly, Glu-110-Asp,and Glu-112-Asp mutants. Other adjuvants (such as a bacterialmonophosphoryl lipid A (MPLA) of various sources (e.g., E. coli,Salmonella minnesota, Salmonella typhimurium, or Shigella flexneri,saponins, or polylactide glycolide (PLGA) microspheres) can also be usedin mucosal administration.

Other adjuvants include cytokines such as interleukins for example IL-1,IL-2 and IL-12, chemokines, for example CXCL10 and CCL5, macrophagestimulating factor, and/or tumor necrosis factor. Other adjuvants thatmay be used include CpG oligonucleotides (Davis. Curr Top MicrobiolImmunol., 247:171-183, 2000).

Oil in water emulsions include squalene; peanut oil; MF59 (WO 90/14387);SAF (Syntex Laboratories, Palo Alto, Calif.); and Ribi™ (RibiImmunochem, Hamilton, Mont.). Oil in water emulsions may be used withimmunostimulating agents such as muramyl peptides (for example,N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP),-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutamyl-L-alanine-2-(1′-2′dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(MTP-PE),N-acetylglucsaminyl-N-acetylmuramyl-L-A-D-isoglu-L-Ala-dipalmitoxypropylamide (DTP-DPP) theramide (TM)), or other bacterial cell wallcomponents.

Adjuvants useful for both mucosal and parenteral immunization includepolyphosphazene (for example, WO 95/2415), DC-chol (3b-(N—(N′,N′-dimethyl aminomethane)-carbamoyl) cholesterol (for example,U.S. Pat. No. 5,283,185 and WO 96/14831) and QS-21 (for example, WO88/9336).

An adjuvant may be coupled to an immunogen for administration. Forexample, a lipid such as palmitic acid, may be coupled directly to oneor more peptides such that the change in conformation of the peptidescomprising the immunogen does not affect the nature of the immuneresponse to the immunogen.

In an embodiment, the composition comprises an antibody describedherein. In another embodiment, the composition comprises an antibodydescribed herein and a diluent. In an embodiment, the composition is asterile composition.

A further aspect includes an antibody complex comprising an antibodydescribed herein and A-beta, optionally A-beta oligomer. The complex maybe in solution or comprised in a tissue, optionally in vitro.

V. Kits

A further aspect relates to a kit comprising i) an antibody and/orbinding fragment thereof, ii) a nucleic acid, iii) peptide or immunogen,iv) composition or v) recombinant cell described herein, comprised in avial such as a sterile vial or other housing and optionally a referenceagent and/or instructions for use thereof.

In an embodiment, the kit further comprises one or more of a collectionvial, standard buffer and detection reagent.

IV. Methods

Included are methods for making the compounds, immunogens and antibodiesdescribed herein.

In particular, provided are methods of making an antibody selective fora conformational epitope of HHQK (SEQ ID NO:1) or related epitopecomprising administering to a subject, optionally a non-human subject, aconformationally restricted compound comprising an epitope sequencedescribed herein, optionally cyclic compound comprising HHQK (SEQ IDNO: 1) or related epitope, and isolating antibody producing cells orantibodies that specifically or selectively bind the cyclic compound andoptionally i) specifically or selectively bind synthetic and/or nativeoligomers and/or that have no or negligible senile plaque binding insitu tissue samples or no or negligible binding to a correspondinglinear peptide. The cyclic compound can for example comprise any of the“epitopes” described herein containing cyclic compounds describedherein.

In an embodiment, the method is for making a monoclonal antibody usingfor example a method as described herein.

In an embodiment, the method is for making a humanized antibody usingfor example a method described herein.

Antibodies produced using a cyclic compound are selected as describedherein and in the Examples such. In an embodiment, the method comprisesisolating antibodies that they specifically or selectively bind cyclicpeptide over linear peptide, are specific for the epitope sequence,specifically bind oligomer and/or lack or negligibly bind plaque in situand/or corresponding linear peptide, optionally using a method describedherein.

A further aspect provides a method of detecting whether a biologicalsample comprises A-beta the method comprising contacting the biologicalsample with an antibody described herein and/or detecting the presenceof any antibody complex. In an embodiment, the method is for detectingwhether a biological sample comprises A-beta wherein at least one of theresidues H, Q, or K is in an alternate conformation than occupied by H,Q and/or K in a non-oligomeric conformation. In an embodiment the methodis for detecting whether the biologic sample comprises oligomericA-beta.

In an embodiment, the method comprises:

a. contacting the biologic sample with an antibody described herein thatis specific and/or selective for A-beta oligomer herein under conditionspermissive to produce an antibody: A-beta oligomer complex; and

b. detecting the presence of any complex;

wherein the presence of detectable complex is indicative that the samplemay contain A-beta oligomer.

In an embodiment, the level of complex formed is compared to a testantibody such as a suitable Ig control or irrelevant antibody.

In an embodiment, the detection is quantitated and the amount of complexproduced is measured. The measurement can for example be relative to astandard.

In an embodiment, the measured amount is compared to a control.

In another embodiment, the method comprises:

(a) contacting a test sample of said subject with an antibody describedherein, under conditions permissive to produce an antibody-antigencomplex;

(b) measuring the amount of the antibody-antigen complex in the testsample; and

(c) comparing the amount of antibody-antigen complex in the test sampleto a control;

wherein detecting antibody-antigen complex in the test sample ascompared to the control indicates that the sample comprises A-beta.

The control can be a sample control (e.g. from a subject without AD, orfrom a subject with a particular form of AD, mild, moderate oradvanced), or be a previous sample from the same subject for monitoringchanges in A-beta oligomer levels in the subject.

In an embodiment, an antibody described herein is used.

In an embodiment, the antibody specifically and/or selectivelyrecognizes a conformation of A-beta comprising HQK or HHQK (SEQ IDNO: 1) or a related conformational epitope and detecting the presence ofantigen: antibody complex is indicative that the sample comprises A-betaoligomer.

In an embodiment, the sample is a biological sample. In an embodiment,the sample comprises brain tissue or an extract thereof and/or CSF. Inan embodiment, the sample comprises whole blood, plasma or serum. In anembodiment, the sample is obtained from a human subject. In anembodiment, the subject is suspected of, at a risk of or has AD.

A number of methods can be used to detect an A-beta: antibody complexand thereby determine A-beta comprising a HHQK (SEQ ID NO: 1) or relatedconformational epitope and/or A-beta oligomers is present in a sampleusing the antibodies described herein, including immunoassays such asflow cytometry, Western blots, ELISA, and immunoprecipitation followedby SDS-PAGE immunocytochemistry.

As described in the Examples surface plasmon resonance technology can beused to assess conformation specific binding. If the antibody is labeledor a detectably labeled secondary antibody specific for the complexantibody is used, the label can be detected. Commonly used reagentsinclude fluorescent emitting and HRP labeled antibodies. In quantitativemethods, the amount of signal produced can be measured by comparison toa standard or control. The measurement can also be relative.

A further aspect includes a method of measuring a level of or imagingA-beta in a subject or tissue, optionally where the A-beta to bemeasured or imaged is oligomeric A-beta. In an embodiment, the methodcomprises administering to a subject at risk or suspected of having orhaving AD, an antibody conjugated to a detectable label; and detectingthe label, optionally quantitatively detecting the label. The label inan embodiment is a positron emitting radionuclide which can for examplebe used in PET imaging.

A further aspect includes a method of inducing an immune response in asubject, comprising administering to the subject a compound, immunogenand/or composition comprising a compound described herein, such as acyclic compound comprising HHQK (SEQ ID NO: 1) or a related epitope; andoptionally isolating cells and/or antibodies that specifically bind thecompound or immunogen administered.

In an embodiment, the immunogen administered comprises a compound ofFIG. 7C.

In an embodiment, the subject is a non-human subject such as a rodent.Antibody producing cells generated are used in an embodiment to producea hybridoma cell line.

It is demonstrated herein that antibodies raised against cyclo(CGHHQKG)(SEQ ID NO: 2), can specifically and/or selectively bind A-betaoligomers and lack A-beta plaque staining. Oligomeric A-beta species arebelieved to be the toxic propagating species in AD. Further as shown inFIG. 19, antibody raised using cyclo(CGHHQKG) (SEQ ID NO: 2) andspecific for oligomers, inhibited A-beta aggregation and A-beta oligomerpropagation. Accordingly, also provided are methods of inhibiting A-betaoligomer propagation, the method comprising contacting a cell or tissueexpressing A-beta with or administering to a subject in need thereof aneffective amount of an A-beta oligomer specific or selective antibodydescribed herein to inhibit A-beta aggregation and/or oligomerpropagation. In vitro the assay can be monitored as described in theExamples.

The antibodies may also be useful for treating AD and/or other A-betaamyloid related diseases. For example, variants of Lewy body dementiaand in inclusion body myositis (a muscle disease) exhibit similarplaques as AD and A-beta can also form aggregates implicated in cerebralamyloid angiopathy. As mentioned, antibodies raised to cyclo(CGHHQKG)(SEQ ID NO: 2) bind oligomeric A-beta which is believed to be atoxigenic species of A-beta in AD and inhibit formation of toxigenicA-beta oligomers.

Accordingly a further aspect is a method of treating AD and/or otherA-beta amyloid related diseases, the method comprising administering toa subject in need thereof i) an effective amount of an antibodydescribed herein, optionally an A-beta oligomer specific or selective ora pharmaceutical composition comprising said antibody; or 2)administering an isolated cyclic compound comprising HHQK (SEQ ID NO: 1)or a related epitope sequence or immunogen or pharmaceutical compositioncomprising said cyclic compound, to a subject in need thereof. In otherembodiments, nucleic acids encoding the antibodies or immunogensdescribed herein can also be administered to the subject, optionallyusing vectors suitable for delivering nucleic acids in a subject.

In an embodiment, a biological sample from the subject to be treated isassessed for the presence or levels of A-beta using an antibodydescribed herein. In an embodiment, a subject with detectable A-betalevels (e.g. A-beta antibody complexes measured in vitro or measured byimaging) is treated with the antibody.

The antibody and immunogens can for example be comprised in apharmaceutical composition as described herein, and formulated forexample in vesicles for improving delivery.

One or more antibodies targeting HHQK (SEQ ID NO: 1) and/or relatedantibodies can be administered in combination. In addition theantibodies disclosed herein can be administered with one or more othertreatments such as a beta-secretase inhibitor or a cholinesteraseinhibitor.

In an embodiment, the antibody is a conformation specific/selectiveantibody, optionally that specifically or selectively binds A-betaoligomer.

Also provided are uses of the compositions, antibodies, isolatedpeptides, immunogens and nucleic acids for treating AD.

The compositions, compounds, antibodies, isolated peptides, immunogensand nucleic acids, vectors etc. described herein can be administered forexample, by parenteral, intravenous, subcutaneous, intramuscular,intracranial, intraventricular, intrathecal, intraorbital, ophthalmic,intraspinal, intracisternal, intraperitoneal, intranasal, aerosol ororal administration.

In certain embodiments, the pharmaceutical composition is administeredsystemically.

In other embodiments, the pharmaceutical composition is administereddirectly to the brain or other portion of the CNS. For example suchmethods include the use of an implantable catheter and a pump, whichwould serve to discharge a pre-determined dose through the catheter tothe infusion site. A person skilled in the art would further recognizethat the catheter may be implanted by surgical techniques that permitvisualization of the catheter so as to position the catheter adjacent tothe desired site of administration or infusion in the brain. Suchtechniques are described in Elsberry et al. U.S. Pat. No. 5,814,014“Techniques of Treating Neurodegenerative Disorders by Brain Infusion”,which is herein incorporated by reference. Also contemplated are methodssuch as those described in US patent application 20060129126 (Kaplittand During “Infusion device and method for infusing material into thebrain of a patient”. Devices for delivering drugs to the brain and otherparts of the CNS are commercially available (eg. SynchroMed® EL InfusionSystem; Medtronic, Minneapolis, Minn.)

In another embodiment, the pharmaceutical composition is administered tothe brain using methods such as modifying the compounds to beadministered to allow receptor-mediated transport across the blood brainbarrier.

Other embodiments contemplate the co-administration of the compositions,compounds, antibodies, isolated peptides, immunogens and nucleic acidsdescribed herein with biologically active molecules known to facilitatethe transport across the blood brain barrier.

Also contemplated in certain embodiments, are methods for administeringthe compositions, compounds, antibodies, isolated peptides, immunogensand nucleic acids described herein across the blood brain barrier suchas those directed at transiently increasing the permeability of theblood brain barrier as described in U.S. Pat. No. 7,012,061 “Method forincreasing the permeability of the blood brain barrier”, hereinincorporated by reference.

The above disclosure generally describes the present application. A morecomplete understanding can be obtained by reference to the followingspecific examples. These examples are described solely for the purposeof illustration and are not intended to limit the scope of theapplication. Changes in form and substitution of equivalents arecontemplated as circumstances might suggest or render expedient.Although specific terms have been employed herein, such terms areintended in a descriptive sense and not for purposes of limitation.

The following non-limiting examples are illustrative of the presentdisclosure:

EXAMPLES Example 1 Collective Coordinates Predictions

A method for predicting misfolded epitopes is provided by a methodreferred to as “Collective Coordinates biasing” which is described inU.S. Patent Application Ser. No. 62/253,044, SYSTEMS AND METHODS FORPREDICTING MISFOLDED PROTEIN EPITOPES BY COLLECTIVE COORDINATE BIASINGfiled Nov. 9, 2015, and is incorporated herein by reference. Asdescribed therein, the method uses molecular-dynamics-based simulationswhich impose a global coordinate bias on a protein (orpeptide-aggregate) to force the protein (or peptide-aggregate) tomisfold and then predict the most likely unfolded regions of thepartially unstructured protein (or peptide aggregate). Biasingsimulations were performed and the solvent accessible surface area(SASA) corresponding to each residue index (compared to that of theinitial structure of the protein under consideration). SASA represents asurface area that is accessible to H₂O. A positive change in SASA(compared to that of the initial structure of the protein underconsideration) may be considered to be indicative of unfolding in theregion of the associated residue index. The method was applied to asingle-chain, and three A-beta strains, each with its own morphology: athree-fold symmetric structure of Aβ-40 peptides (or monomers) (PDBentry 2M4J), a two-fold symmetric structure of Aβ-40 monomers (PDB entry2LMN), and a single-chain, parallel in-register (e.g. a repeated betasheet where the residues from one chain interact with the same residuesfrom the neighboring chains) structure of Aβ-42 monomers (PDB entry2MXU).

Simulations were performed for each initial structure using thecollective coordinates method as described in U.S. Patent ApplicationSer. No. 62/253,044, SYSTEMS AND METHODS FOR PREDICTING MISFOLDEDPROTEIN EPITOPES BY COLLECTIVE COORDINATE BIASING and the CHARMMforce-field parameters described in: K. Vanommeslaeghe, E. Hatcher, C.Acharya, S. Kundu, S. Zhong, J. Shim, E. Darian, O. Guvench, P. Lopes,I. Vorobyov, and A. D. Mackerell. Charmm general force field: A forcefield for drug-like molecules compatible with the charmm all-atomadditive biological force fields. Journal of Computational Chemistry,31(4):671-690, 2010; and P. Bjelkmar, P. Larsson, M. A. Cuendet, B.Hess, and E. Lindahl. Implementation of the CHARMM force field inGROMACS: analysis of protein stability effects from correlation maps,virtual interaction sites, and water models. J. Chem. Theo. Comp.,6:459-466, 2010, both of which are hereby incorporated herein byreference, with TIP3P water.

Epitopes predicted using this method are described in Example 2.

G  o Model Method for Predicting A-Beta Oligomer Specific Epitopes

A second epitope prediction model is based on the free energy landscapeof partial protein unfolding from the native state. The native state istaken to be an experimentally-derived fibril structure. When the proteinis partially unfolded from the native state by a given amount of primarysequence, epitope candidates are contiguous sequence segments that costthe least free energy to disorder. The free energy of a given proteinconformation arises from several contributions, including conformationalentropy and solvation of polar functional groups that favor the unfoldedstate, as well as the loss of electrostatic and van der Waalsintra-protein interactions that enthalpically stabilize the nativestate.

A. Gō-Like Model of Protein Partially Unfolding Landscape

An approximate model to account for the free energetic changes that takeplace during unfolding assigns a fixed energy to all contacts in thenative state, where a contact is defined as a pair of heavy(non-hydrogen) atoms within a fixed cut-off distance r_(cutoff). Gō-likemodels have been successfully implemented in previous studies of proteinfolding. The Gō-like model isolates the effects arising from thetopology of native protein interactions, and in practice the unfoldingfree energy landscape can be readily calculated from a single nativestate structure.

The total free energy cost of unfolding a segment depends on the numberof interactions to be disrupted, together with the conformationalentropy term of the unfolded region.

In the following equations, lowercase variables refer to atoms, whileupper case variables refer to residues. Let T be the set of all residuesin the protein, U be the set of residues unfolded in the protein, and Fbe the subset of residues folded in the protein (thus T=U∪F). Theunfolding mechanism at high degrees of nativeness consists of multiplecontiguous strands of disordered residues. Here the approximation of asingle contiguous unfolded strand was adopted, and the free energy costto disorder this contiguous strand was calculated.

The total free energy change ΔF_(Gō)(U) for unfolding the set ofresidues U is

ΔF _(Gō)(U)=ΔE _(Gō)(U)−TΔS _(Gō)(U)  (1)

The unfolding enthalpy function ΔE_(Gō)(U) is given by the number ofinteractions disrupted by unfolding of the set of U residues:

$\begin{matrix}{{\Delta \; {E_{G\overset{\_}{o}}()}} = {a{\sum\limits_{{{{Atoms}\mspace{14mu} i} \in },{j \in }}^{i > j}{\Theta \left( {r_{cutoff} - {{r_{i} - r_{j}}}} \right)}}}} & (2)\end{matrix}$

In Equation 2, the sum on i, j is over all unique pairs of heavy atomsthat have either one or both atoms in the unfolded region, r_(i) andr_(j) are the coordinates of atoms i and j, r_(cutoff) (taken to be 4.8Å) is the interaction distance cut-off. Θ(x) is the Heaviside functiondefined by Θ(x)=1 if x is positive and 0 otherwise. The energy percontact a may be chosen to recapitulate the overall experimentalstability ΔF_(Exp)(U)|_(U=T) on completely unfolding the protein at roomtemperature:

$\begin{matrix}{a = \frac{{\Delta \; {F_{Exp}()}}_{ = }{{{+ T}\; \Delta \; {S_{G\overset{\_}{o}}()}}_{ = }}}{\Sigma_{i,{j \in }}^{i > j}{\Theta \left( {r_{cutoff} - {{r_{i} - r_{j}}}} \right)}}} & (3)\end{matrix}$

The results do not depend on this value; it merely sets the overallglobal energy scale in the problem. In the present model, this freeenergy was taken to be a constant number equal to 4.6 kcal/mol. Thisvalue is not a primary concern as it is the relative free energy costfor the different regions of the same protein that is sought to bedisordered in the method of epitope prediction.

The calculation of the unfolding entropy term ΔS_(Gō)(U) is discussed inB below.

B. Entropy Calculation

The number of microstates accessible to the protein in the unfoldedstate is much greater than the number accessible in the native state, sothere is a favorable gain of conformational entropy on unfolding. Thetotal entropy of the unfolding segment U by summing over all theresidues K in the unfolded region is calculated

$\begin{matrix}{{\Delta \; {S_{G\overset{\_}{o}}()}} = {\sum\limits_{K \in }\left( {{\Delta \; S_{{bb},K}} + {\left( {1 - \frac{A_{,K}}{A_{,K}}} \right)\Delta \; S_{{{bu}\rightarrow{ex}},K}} + {\Delta \; S_{{{ex}\rightarrow{sol}},K}}} \right)}} & (4)\end{matrix}$

where ΔS_(bb,K), ΔS_(bu→ex,K), ΔS_(ex→sol,K) are the threeconformational entropic components of residue K as listed in reference[3]: ΔS_(bb,K) is the backbone entropy change from native state tounfolded state, ΔS_(bu→ex,K) is the entropy change for side-chain fromburied inside protein to the surface of the protein, and, andΔS_(ex→sol,K) is the entropy obtained for the side-chain from thesurface to the solution.

A correction is applied to the unfolded state conformational entropies,since in the single sequence approximation the end points of thepartially unfolded strand are fixed in their positions in the nativestructure. This means that there is a loop entropy penalty to be paidfor constraining the ends in the partially unfolded structure, which isnot present in the fully unfolded state

ΔS _(return) =−k _(B) ln(f _(w)(R|N)Δτ.  (5)

Here f_(w)(R|N)Δτ is found by calculating the probability an idealrandom walk returns to a box of volume Δτ centered at position R after Nsteps, without penetrating back into the protein during the walk. Forstrand lengths shorter than about n≈20 residues, the size of the meltedstrand is much smaller than the protein diameter and the steric excludedvolume of the protein is well treated as an impenetrable plane. Thenumber of polymeric states of the melted strand must be multiplied bythe fraction of random walks that travel from an origin on the surfaceof the protein to a location where the melted polymer re-enters theprotein without touching or crossing the impenetrable plane. The abovefraction of states can be written in the following form:

$\begin{matrix}{{f_{w}\left( {RN} \right)} = {\frac{a}{N^{5\text{/}2}}{\exp \left( {{- \frac{3R^{2}}{2{Nl}^{2}}} - \frac{N^{2}V_{c}}{2R^{3}}} \right)}}} & (6)\end{matrix}$

where R is the end to end distance between the exit and entrancelocations, N is the number of residues of the melted region, and a, I,V_(c) are parameters determined by fitting to unfolded polypeptidesimulations. The parameter I is the effective arc length between twoC_(a) atoms, and V_(c) is the average excluded volumes for each residue.By fitting the Equation 6 into the simulation results, the values of theparameters a=0.0217, I=4.867, V_(c)=3.291 are obtained. This entropypenalty is general and independent of the sequence.

Disulfide bonds require additional consideration in the loop entropyterm since they further restrict the motion of the unfolded segment.When present, the disulfide is treated as an additional node throughwhich the loop must pass, in effect dividing the full loop into twosmaller loops both subject to the boundary conditions described above.

C. Epitope Prediction from Free Energy Landscape

Once the free energy landscape of partially unfolding the protein isobtained, a variable energy threshold Eth is applied, and the segmentsthat contains no fewer than 3 amino acids and with free energy costbelow the threshold are predicted as epitope candidates. The predictionis stable with respect to varying the threshold value Eth.

Epitopes predicted using this method are described in Example 2.

Example 2

I. Conformation Specific Epitopes

This disclosure pertains to antibodies that may be selective foroligomeric A-beta peptide and particularly to toxic oligomers of Aβpeptide, a species of misfolded protein whose prion-like propagation andinterference with synaptic vesicles are believed to be responsible forthe synaptic dysfunction and cognitive decline that occurs inAlzheimer's disease (AD). Aβ is a peptide of length 36-43 amino acidsthat results from the cleavage of amyloid precursor protein (APP) bygamma secretase. In AD patients, it is present in monomers, fibrils, andin soluble oligomers. Aβ is the main component of the amyloid plaquesfound in the brains of AD patients.

In monomer form, Aβ exists as an unstructured polypeptide chain. Infibril form, Aβ can aggregate into distinct morphologies, often referredto as strains. Several of these structures have been determined bysolid-state NMR—some fibril structures have been obtained from in vitrostudies, and others obtained by seeding fibrils using amyloid plaquestaken from AD patients.

The oligomer is suggested to be a toxic and propagative species of thepeptide, recruiting and converting monomeric Aβ to oligomers, andeventually fibrils.

A prerequisite for the generation of oligomer-specific antibodies is theidentification of targets on Aβ peptide that are not present on eitherthe monomer or fibril. These oligomer-specific epitopes would not differin primary sequence from the corresponding segment in monomer or fibril,however they would be conformationally distinct in the context of theoligomer. That is, they would present a distinct conformation in theoligomer that would not be present in the monomer or fibril.

The structure of the oligomer has not been determined to date, moreover,NMR evidence indicates that the oligomer exists not in a singlewell-defined structure, but in a conformationally-plastic, malleablestructural ensemble with limited regularity. Moreover, the concentrationof oligomer species is far below either that of the monomer or fibril(estimates vary but on the order of 1000-fold below or more), makingthis target elusive.

Antibodies directed either against contiguous strands of primarysequence (e.g., linear sequence), or against fibril structures, maysuffer from several problems limiting their efficacy. Antibodies raisedto linear peptide regions tend not to be selective for oligomer, andthus bind to monomer as well. Because the concentration of monomer issubstantially higher than that of oligomer, such antibody therapeuticsmay suffer from “target distraction”, primarily binding to monomer andpromoting clearance of functional Aβ, rather than selectively targetingand clearing oligomeric species. Antibodies raised to amyloid inclusionsbind primarily to fibril, and have resulted in amyloid related imagingabnormalities (ARIA), including signal changes thought to representvasogenic edema and/or microhemorrhages.

To develop antibodies selective for oligomeric forms of Aβ, a regionthat may be disrupted in the fibril was identified. Without wishing tobe bound to theory, it was hypothesized that disruptions in the contextof the fibril may be exposed as well on the surface of the oligomer. Onoligomers however, these sequence regions may be exposed inconformations distinct from either that of the monomer and/or that ofthe fibril. For example, being on the surface, they may be exposed inturn regions that have higher curvature, higher exposed surface area,different dihedral angle distribution and/or overall differentconformational geometry as determined by structural alignment than thecorresponding quantities exhibit in either the fibril or the monomer(e.g. linear peptide).

Cyclic compounds comprising HHQK (SEQ ID NO: 1) are described herein andshown in FIG. 7 Panel C. The cyclic compounds have been designed tosatisfy one or more of the above criteria.

A potential benefit of identifying regions prone to disruption in thefibril is that it may identify regions involved in secondary nucleationprocesses where fibrils may act as a catalytic substrate to nucleateoligomers from monomers [3]. Regions of fibril with exposed side chainsmay be more likely to engage in aberrant interactions with nearbymonomer, facilitating the accretion of monomers; such accreted monomerswould then experience an environment of effectively increasedconcentration at or near the surface of the fibril, and thus be morelikely to form multimeric aggregates including oligomers. Aged ordamaged fibril with exposed regions of Aβ may enhance the production oftoxic oligomer, and that antibodies directed against these disorderedregions on the fibril could be effective in blocking such propagativemechanisms.

II. Collective Coordinates and Promis Gō Predictions

The epitope HHQK (SEQ ID NO: 1) emerges as a predicted epitope fromstrain 2MXU from the collective coordinates approach, and for strain and2M4J from the Promis Gō approaches described in Example 1 as shown inFIG. 1.

In Panel A, the graph represents the epitope predictions arising fromthe partially-disordered fibril. The HHQK (SEQ ID NO: 1) epitope emergesas a prediction for PDB structure 2MXU (FIG. 1 (Panel A, left) whileHQKL (SEQ ID NO: 20) emerges as a prediction for PDB structure 2M4J(Panel A, right). (FIG. 1 Panel B), HHQK (SEQ ID NO: 1) emerges as anepitope prediction of the ProMis algorithm for chain C of structure2M4J. (Panel C) HHQK (SEQ ID NO: 1) emerges as an epitope for chains G,H, I of 2M4J. The unfolding landscape appears similar for all 3 chainsdue to the 3-fold symmetry of the structure. (Panel D) HHQK (SEQ IDNO: 1) emerges as an epitope for chains L of structure 2MXU. Theoverlapping epitope HQKL (SEQ ID NO: 20) also emerges as a predictedepitope using Collective Coordinates from strain 2M4J (FIG. 1A).

III. Curvature of the Cyclic Peptide

The curvature of the cyclic peptide as a function of residue index wascompared to the curvature of the linear peptide and the fibril.

Curvature values for all residues in the peptide are obtained afteraveraging over the respective equilibrium ensembles. A point (x, y) inthe linear, cyclic, or fibril-2M4J plots of Panels A, B, or C of FIG. 2corresponds to the curvature of native residues 13-16, HHQK (SEQ IDNO:1); residues outside this range in Panels A and B, i.e. 12 in PanelA, and 11, 12, and 17 in Panel B, correspond to non-native residuespresent in the linear and cyclic constructs respectively. Convergence isdemonstrated by averaging over ensembles from 10 ns to increasing times72 ns, 134 ns, 196 ns, and 258 ns. Panel G shows the converged values ofthe curvature for the linear and cyclic peptides along with thecurvature in the fibril. Interestingly, the curvature of Q15 in thecyclic peptide is substantially lower than that in either the linearpeptide or fibril. K16 also has a significantly lower curvature in thecyclic peptide than the linear peptide, and comparable to but stilllower than the curvature in the fibril.

The curvature profiles of the cyclic and linear peptide CGHHQKG (SEQ IDNO: 2), along with the curvature profile of the fibril 2M4J, are shownin FIG. 2G. As shown therein, residue 16K has a different curvature thanthe linear peptide, but a similar albeit still lower curvature comparedto the fibril. Perhaps surprisingly, the glutamine residue 15Q hassignificantly lower curvature in the cyclic peptide compared to thecurvature of 15Q in either the linear peptide or the fibril. Adiscrepancy in curvature is a metric for the discrepancy in antigenicprofiles between the cyclic peptide and other conformational forms.

FIG. 2A plots the curvature for linear CGHHQKG (SEQ ID NO:2) as obtainedfrom different equilibrium simulation times. The legend shows severalcurves that start from 10 ns and continue to either 72 ns, 134 ns, 196ns, or 258 ns. As simulation time is increased, the curvature valuesconverge to the values reported above and in Table 1. Similar studiesare shown in FIG. 2B for the cyclic peptide and FIG. 2C for the fibril.Panels D, E, and F show the convergence in the sum of the curvaturevalues as a function of simulation time, for the linear, cyclic, andfibril conformations respectively. The degree of convergence indicatesthat the error bars are approximately 0.007 radian for the cyclicpeptide, 0.011 radian for the linear peptide, and 0.005 radian for thefibril. It was observed that the curvature values fully converged afterabout 200 ns for the linear ensemble, about 150 ns for the cyclicensemble, and about 20 ns for the fibril ensemble. The average curvatureas a function of residue index for CGHHQKG (SEQ ID No: 2) is shown inPanel G where the linear peptide is in solid dark grey, the cyclicpeptide in solid light grey and the fibril in dotted line. Numericalvalues of the curvature for residues 13H, 14H, 15Q, and 16K are given inTable 1. The curvature for H13 in the cyclic peptide is similar to thelinear peptide, and higher than the fibril. The curvature for H14 in thecyclic peptide is slightly less than that in the linear peptide, butstill higher than that in the fibril. The curvature for Q15 issubstantially less than the curvature in either the linear peptide orthe fibril; the curvature for K16 is also substantially less then tocurvature in the linear peptide, and comparable but still less than thecurvature in the fibril.

For the plots in FIGS. 1-10 discussed herein, the data are obtained fromequilibrium simulations in explicit solvent (TIP3P) using the Charmm27force field. The simulation time and number of configurations for eachensemble are as follows. Cyclic peptide ensemble: simulation time 300ns, containing 10000 frames; linear peptide ensemble: simulation time300 ns, containing 10000 frames; 2M4J ensemble: 60 ns, containing 10000frames.

Because the curvature of the cyclic epitope has a different profile thaneither the linear peptide or fibril, it is expected that thecorresponding stretch of amino acids on an oligomer containing theseresidues would have a backbone orientation that is distinct from that inthe fibril or monomer. However the degree of curvature would not beunphysical—values of curvature characterizing the cyclic peptide areobtained in several residues for the unconstrained linear peptide.

Based on FIG. 2, the curvature values of 13H, 14H, 15Q, and 16K areshown in Table 1 for the linear, cyclic and fibril (2M4J) peptides.

TABLE 1 Curvature value by residue Linear cyclic 2M4J 13H 1.46 1.49 1.1214H 1.47 1.37 1.12 15Q 1.41 0.73 0.99 16K 1.37 1.04 1.15

IV. Dihedral Angle Distributions

Further computational support for the identification of anoligomer-selective epitope, is provided by both the side chain dihedralangle distributions, and the Ramachandran, ϕ and ψ distributions for thebackbone dihedral angles in the cyclic peptide, a proxy for an exposedepitope in the oligomer. Some angles have substantially differentdistributions than the corresponding distributions in either the fibrilor monomer.

The side-chain and backbone dihedral distributions were examined forfour residues 13H, 14H, 15Q and 16K. Percent overlap of distributione.g. “linear” in distribution “cyclic” is obtained by dividing theangles into elements of 5°, then decreasing a cutoff in probabilityamplitude from infinity, until 90% of the cyclic distribution is abovethe cutoff, and 10% remains below. This defines one or more regions inthe allowable angles. Percent of the linear distribution within thisregion was then found. The recipe is non-reciprocal and generally yieldsdifferent numbers between pairs of distributions.

As shown in FIG. 3, for residue 13H, dihedrals C-CA-N-HN and O-C-CA-CBclearly distinguish both linear and cyclic peptides of HHQK (SEQ IDNO: 1) from the corresponding dihedral angles in the fibril. For residue14H, dihedral angles C-CA-N-HN and O-C-CA-CB clearly distinguish thecyclic dihedral angle distribution from the corresponding distributionsin either the linear or fibril ensembles. Likewise, for residue 15Q,dihedral angles C-CA-N-HN and O-C-CA-CB clearly distinguish the cyclicdihedral angle distribution from the corresponding distributions ineither the linear or fibril ensembles. For residue 16K, dihedral angleO-C-CA-CB distinguishes the cyclic peptide from either the linear orfibril ensembles, and dihedral angle C-CA-N-HN distinguishes both cyclicand linear peptides from the fibril. According to FIG. 5B, the backboneRamachandran angles ϕ and ψ of 13H distinguish the linear and cyclicpeptides from the fibril, but not from each other. For 14H, FIG. 5Cshows that Ramachandran angles ϕ and ψ of the cyclic peptide are bothdistinct from either the linear or fibril ensembles. Likewise for 15Qand 16K, FIGS. 5D and E show that the Ramachandran angles ϕ and ψ of thecyclic peptide are distinct from those in either the linear or fibrilensembles.

From the dihedral distributions shown in FIG. 3, the probability thatthe linear peptide occupies a dihedral within the range of almost all(90%) of the cyclic peptide dihedral angles is as follows for thedihedral angles: 14H:C-CA-N-HN, 14%; 15Q:O-C-CA-CB, 11%; 16K:O-C-CA-CB,28%. All overlap probabilities are given in Table 2.

It is important to note that the accumulation of relatively smalldifferences in individual dihedral angles can result in a large andsignificant difference in global conformation of the peptide, and thussignificant deviations in the structural alignment, as described furtherin Example VIII below.

The probability that the peptide in the context of the fibril occupies adihedral within the range of almost all (90%) of the cyclic peptidedihedral angles is as follows for the dihedral angles of: 13H:O-C-CA-CB,0%; 14H:O-C-CA-CB, 30%; 15Q:O-C-CA-CB, 45%; 16K:C-CA-N-HN, 6%. For alloverlap probabilities see Table 2. Note again that the accumulation ofrelatively small differences in individual dihedral angles can result ina large and significant difference in global conformation of thepeptide, and thus significant deviations in the structural alignment, asdescribed further in Example VIII below.

Based on FIG. 3, Table 2 shows the percent overlap of dihedral angledistributions for backbone and side-chain angles of residues H13, H14,Q15, and K16 in linear, cyclic and fibril (2M4J) forms relative to eachother. E.g. Column 2 shows the percentage overlap between a givendihedral angle in the linear peptide and the same angle in the cyclicform.

TABLE 2 Percent overlap of dihedral angle distribution. Linear 2M4JCyclic 2M4J Linear Cyclic in in in in in in cyclic cyclic linear linear2M4J 2M4J 13H:C-CA-CB- 91% 97% 87% 94% 30% 42% CG 13H:C-CA-N- 83% 54%97% 69% 57% 73% HN 13H:CA-CB- 94% 85% 79% 85% 72% 47% CG-CD2 13H:O-C-CA-79%  0% 96%  0%  4%  0% CB 14H:C-CA-CB- 93% 89% 78% 81% 27% 51% CG14H:C-CA-N- 13% 83% 17% 49% 30% 78% HN 14H:CA-CB- 90% 83% 81% 84% 86%73% CG-CD2 14H:O-C-CA- 47% 30% 68% 14% 17% 77% CB 15Q:C-CA- 89% 87% 84%89% 91% 91% CB-CG 15Q:C-CA-N- 48% 62% 28% 71% 96% 55% HN 15Q:NE2-CD- 90%89% 90% 88% 90% 92% CG-CB 15Q:O-C-CA- 11% 45% 69% 50% 13% 72% CB16K:C-CA-CB- 86% 80% 86% 92% 59% 30% CG 16K:C-CA-N- 68%  6% 89% 83% 26% 4% HN 16K:O-C-CA- 28% 28% 79% 45%  9% 10% CB

According to the above analysis of side chain and backbone dihedralangle distributions, residues Q15 and K16 show significant discrepanciesfrom the linear peptide and fibril ensembles. By these metrics, Q15 andK16 may be key residues on the epitope conferring conformationalselectivity. Residue H14 shows smaller discrepancies, but may assist inconferring conformational selectivity.

Based on the data shown in FIG. 3, Table 3 lists the peak values of thedihedral angle distributions, for those dihedral angles whosedistributions that show significant differences between the cyclicpeptide and other species. Column 1 in Table 3 is the specific dihedralconsidered, column 2 is the peak value of the dihedral distribution forthat angle in the context of the cyclic peptide CGHHQKG (SEQ ID NO: 2),column 3 is the peak value of the dihedral distribution for that anglein the context of the linear peptide CGHHQKG (SEQ ID NO: 2), column 4 isthe peak value of the dihedral distribution for the peptide HHQK (SEQ IDNO: 1) in the context of the fibril structure 2M4J, and column 5 is thedifference of the peak values of the dihedral distributions for thelinear and cyclic peptides.

TABLE 3 Peak Values of the Dihedral Angle Distributions Dihedral anglecyclic linear 2M4J cyclic-linear 13H:C-CA-CB-CG 178 −63 178 24013H:C-CA-N-HN 113 118 73 −5 13H:CA-CB-CG-CD2 113 103 93 10 13H:O-C-CA-CB−98 −93 63 −5 14H:C-CA-CB-CG −58 −63 178 5 14H:C-CA-N-HN 48 113 73 −6514H:CA-CB-CG-CD2 108 118 98 −10 14H:O-C-CA-CB −43 −93 58 5015Q:C-CA-CB-CG −63 178 178 −240 15Q:C-CA-N-HN 33 113 68 −8015Q:NE2-CD-CG-CB −73 78 98 −150 15Q:O-C-CA-CB 108 −93 73 20016K:C-CA-CB-CG −58 173 63 −230 16K:C-CA-N-HN 123 118 68 5 16K:O-C-CA-CB108 −98 68 205

V. Entropy of the Side Chains

The side chain entropy of a residue may be approximately calculated from

${{S\text{/}k}_{\mathcal{B}}} = {- {\sum\limits_{i}{\int{d\; \varphi_{i}{p\left( \varphi_{i} \right)}\mspace{14mu} \ln \mspace{14mu} {{p\left( \varphi_{i} \right)}.}}}}}$

Where the sum is over all independent dihedral angles in a particularresidue's side chain, and p(ϕ_(i)) is the dihedral angle distribution,as analyzed above.

Dissection of Entropy of Residue Side-Chain Moieties

The entropy of each dihedral angle was investigated for 13H, 14H, 15Qand 16K. The entropy of the dihedral angles for each of the residues isplotted in FIG. 4 Panels A-D. The entropy for several dihedrals of 15Qand 16K is reduced relative to the linear form, indicating a restrictedpose for those angles in a conformation that tends to be distinct fromthe linear form and thus likely the monomer. Panel E plots the totalside chain entropy (not including Ramachandran backbone angles) forresidues 13H, 14H, 15Q and 16K, relative to the entropy of the fibril,e.g. ΔS for the cyclic peptide is S(cyclic)−S(fibril). This shows thatentropy is increased relative to the fibril for cyclic peptide, and isincreased for 13H, 15Q, and 16K for the linear peptide, but reducedrelative to the fibril for 14H. As well, the cyclic peptide is seen tohave less entropy than the linear peptide. Panel F plots the total sidechain plus Ramachandran backbone entropy for residues 13H, 14H, 15Q and16K relative to the entropy of the fibril. This shows again that entropyis increased relative to the fibril for 15Q and 16K, but that the cyclicpeptide has less entropy than the linear peptide for those residues, andso is more strongly constrained. On the other hand, H14 shows lessentropy than the fibril for both cyclic and linear forms, with a linearform showing the least entropy of all. This means that fibrilconstraints on other parts of the peptide actually increase the entropyof H14. Panel G again plots the total conformational entropy forresidues 13H, 14H, 15Q and 16K, now relative to the entropy of thelinear monomer. This shows reduced conformational entropy in the cyclicpeptide relative to the linear peptide for residues H13, Q15, and K16.The total reduction in entropy is only about 1 kB however. Theprobability to be in this slightly restricted set of conformations isless than exp(−ΔS)≈0.37 however, because even though the cyclicconformations have substantial entropy, they correspond to a distinctnonoverlapping distribution of conformations, as described above in thecontext of dihedral angle overlap and below in example VIII in thecontext of structural overlap.

The cyclic peptide is more rigid than the linear peptide for residues15Q and 16K. The entropies are all comparable for H13, and the entropyof the cyclic peptide is increased from the linear for H14; for H14,both cyclic and linear entropies are less than the entropy in thefibril, indicating that interestingly, energetic constraints in thefibril result in the increase in entropy of H14. The entropy of thecyclic peptide is reduced from the entropy of the linear peptide byabout 1 kB. Lower side chain conformational entropy in the cyclicpeptide supports a more well-defined conformational pose that could aidin conferring selectivity.

VI. Ramachandran Angles

The backbone orientation that the epitope exposes to an antibody differsdepending on whether the peptide is in the linear, cyclic, or fibrilform. This discrepancy can be quantified by plotting the Ramachandranangles phi and psi (or ϕ and ψ), along the backbone, for each residue13H, 14H, 15Q, and 16K in both the linear and cyclic peptides. FIG. 5plots the phi and psi angles sampled in equilibrium simulations, forresidues 13H, 14H, 15Q, and 16K in both linear and cyclic peptidesconsisting of sequence CGHHQKG (SEQ ID No: 2), as well as HHQK (SEQ IDNO: 1) in the context of the fibril structure 2M4J. From FIG. 5 panel B,it can be seen that the distributions of backbone dihedral angles for14H, 15Q, and 16K in the cyclic peptide are different from thedistributions of dihedral angles sampled for either the linear peptideor fibril.

The probabilities of the Ramachandran angles of the residue 14H in thelinear form overlapping with 90% of the Ramachandran angles in thecyclic form is 10%; the corresponding overlap for the fibril with thecyclic is 23%. The probabilities of the Ramachandran angles of theresidue 13H linear form overlapping with the cyclic form it Is muchhigher, 76%. However there is negligible probability of the fibril formoverlapping with 90% of the Ramachandran angles in the cyclic form (0%).The corresponding probabilities for 15Q are 10% and 28% respectively.The corresponding probabilities for 16K are 32% and 1% respectively. SeeTable 4.

TABLE 4 Overlap probabilities for Ramachandran angles cyclic in 2M4J inlinear in 2M4J in Linear in cyclic in linear linear cyclic cyclic 2M4J2M4J 13H 88% 0.30% 76%  0     3%   0% 14H 24%   13% 10% 23% 13%   67%15Q 66%   35% 10% 28% 8%   26% 16K 58%   48% 32%  1%  5% 0.60%

Table 5 gives the peak (most-likely) values of the Ramachandran ϕ,ψangles plotted in FIG. 5 for residues 13H, 14H, 15Q, and 16K. Themost-likely Ramachandran phi and psi values are different between thecyclic and linear peptides for residues H14, Q15, and K16. For H14, thepeak values in the cyclic distribution are (−65,−45) degrees, while thepeak values in the linear and fibril distributions are at (−145,20) and(−115,115),(−115,15) respectively. The differences between these phi andpsi values cyclic-linear are 80, and 65 degrees, and the differencesbetween the phi and psi values cyclic-fibril are 50, 160, and 60degrees. The Ramachandran values are substantially different between thelinear and cyclic peptides, and fibril and cyclic peptides.

Table 5 also describes differences in phi psi angles for Q15 and K16.The differences delta(phi) for Q15 between cyclic and linear is 95degrees; for delta(psi) for Q15, the difference between cyclic andlinear is 200 degrees; between cyclic and fibril it is up to 45 degrees.For K16 the difference delta(phi) is about 190 degrees between cyclicand linear; the difference delta(psi) is about 55 degrees between cyclicand fibril. The difference in many of these peak dihedral angle valuesimplies that antibodies selected for the cyclic epitope conformationwill likely have lower affinity for the linear and fibril epitopes.

The peak values (most likely values) of the Ramachandran backbone ϕ,ψdistributions for 13H, 14H, 15Q, and 16K are given in Table 5. The firstcolumn in Table 5 gives the residue considered, which manifests twoangles, phi and psi, indicated in parenthesis. The 2^(nd) columnindicates the peak values of the Ramachandran phi/psi angles in thecontext of the linear peptide CGHHQKG (SEQ ID No:2), while the 3^(rd)column indicates the peak values of the Ramachandran phi/psi angles inthe context of the cyclic peptide CGHHQKG (SEQ ID No:2), and the lastcolumn indicates the peak values of the Ramachandran phi/psi angles inthe context of the fibril structure 2M4J.

TABLE 5 Peak values of distributions of backbone phi/psi angles Peakvalues of distributions of 13-16 HHQK (SEQ ID NO: 1) backbone phi/psiangles linear cyclic fibril 13H (−60, −35) (−65, −40) (−120, 115) 14H(−65, −45) (−145, 20) (−115, 115)(−115, 15) 15Q (−65, −40) (−160, 160)(−65, 145) (−130, 125) 16K (−65, −45) (−65, 145) (−120, 125)

VII. Solubility and Antigenicity of the Epitope

FIG. 6 Panel A plots the intrinsic solubility of each amino acid in thecontext of the native sequence of A-beta peptide. FIG. 6 Panel B plotsthe mean solvent accessible surface area (SASA) of each residue in theequilibrium ensemble of the cyclic peptide, the linear peptide, and thefibril. This shows that the SASA of residues HHQK (SEQ ID NO: 1) in thecyclic peptide is increased over the fibril, and as well, the SASA ismodestly increased over the linear peptide, indicating more surfacewould be exposed and thus accessible to antibody binding. The increasein exposure is most significant for residue K16, which shows the largestincrease in SASA over the linear peptide.

FIG. 6C shows the SASA weighted by the solubility given in FIG. 6A. Theweighting factor is given by the solubility of the given residue minusthe minimum solubility in A-beta fibril, divided by the standarddeviation of the solubilities in the fibril. Weighted solubilities areplotted for each residue in the cyclic, linear, and fibril ensembles.FIG. 6D shows the change and weighted solubility with respect to thefibril for both the cyclic and linear peptides. Together these plotsshow that residue K16 is significantly solvent exposed and accessiblefor binding, and that residues H13 and H14 will also tend to be solventexposed for binding.

There is no definitive evidence as to which residue will have the mostlikelihood of differential exposure and availability for antibodybinding, as compared to those residues in the conformation of HHQK (SEQID NO: 1) in the fibril structure, however the plots in FIG. 6 show thatall residues including histidines H13 and H14 should be available forantibody binding.

VIII. The Ensemble of Cyclic Peptide Conformations Clusters Differentlythan the Ensemble of Either Linear or Fibril Conformations

Definitive evidence that the sequence HHQK (SEQ ID No: 1) displays adifferent conformation in the context of the cyclic peptide than in thelinear peptide can be seen by using standard structural alignmentmetrics between conformations, and then implementing clusteringanalysis. Equilibrium ensembles of conformations are obtained for thelinear and cyclic peptides CGHHQKG (SEQ ID No: 2), as well as thefull-length fibril in the structure corresponding to PDB ID 2M4J.Snapshots of conformations from these ensembles for residues HHQK (SEQID NO: 1) are collected and then structurally aligned to the centroidsof the largest cluster of the cyclic peptide ensemble, the largestcluster of the linear peptide ensemble, and the largest cluster of HHQK(SEQ ID NO: 1) in the fibril ensemble; the three values of the root meansquared deviation (RMSD) are then recorded and plotted. The clusteringis performed here by the maxcluster algorithm(http://www.sbq.bio.ic.ac.uk/maxcluster). The 3 corresponding RMSDvalues for the linear, cyclic, and fibril ensembles are plotted as a3-dimensional scatter plot in FIG. 9. FIG. 9 panels A, B, C show 3different views of the 3-dimensional scatter plot.

Table 6 shows the percentage overlap of the RMSD scatter plot of thelinear, cyclic and fibril (2M4J) peptide conformations. Column 1 showsthe percentage overlap from the linear form to the cyclic form is quitesmall, only 7%.

TABLE 6 Percentage overlap of RMSD clustering linear 2M4J cyclic 2M4Jlinear Cyclic linear linear linear in in in in in in in in in cycliccyclic linear linear 2M4J 2M4J 2LMP 2MXU 2LMN 7% 0 32% 0.6% 0.03% 0 0%0.35% 0.01%

It is evident from FIG. 9 and Table 6 that the 3 ensembles clusterdifferently from each other. In particular, the cyclic peptidestructural ensemble is distinct from either the linear or fibrilensembles, implying that antibodies specific to the cyclic peptideepitope may have low affinity to the conformations presented in thelinear or fibril ensembles. An antibody raised to the cyclic peptidecould be conformationally selective and preferentially bind oligomericforms over either the linear or fibril conformations of A-beta. Thedistinction between the ensembles occurs in spite of the overlap betweenseveral side chain and backbone dihedral angle distributions; thenumerous often small differentiating features described above lead toglobally different conformational distributions.

The overlap between the ensembles was calculated as follows. Thefraction (percent) of the linear ensemble that overlaps with the cyclicensemble is obtained by first dividing the volume of this 3-dimensionalRMSD space up into cubic elements of length 0.1 Angstrom. Then a “cutoffdensity” of points in the cyclic distribution is found such that thecubes with cyclic distribution density equal to or higher than thecutoff density contain 90% of the cyclic distribution. This defines avolume (which may be discontiguous) that gives the characteristic volumecontaining the cyclic distribution and removes any artifacts due tooutliers. Then the fraction of points from the linear distribution thatare within this region is found. With this method, it is possible tofind the overlapping percentages for fibril in linear, cyclic in linear,etc.

The numeric overlapping percentage obtained by the above method is givenin Table 6. In particular, the cyclic peptide and the fibril peptide2M4J have 0% overlap. By the above recipe, the overlap of the lineardistribution within the cyclic distribution is 7%, meaning that thelinear peptide is sampling states distinct from the cyclic conformationapproximately 93% of the time.

FIG. 9 Panel D shows the percent overlap of the linear ensemble with 90%of the fibril ensemble, as described in the text and Panel E shows thepercent overlap of the linear ensemble with 90% of the cyclic ensemble.This number is particularly important because it indicates thelikelihood of the linear peptide adopting a confirmation consistent withthe cyclic peptide. Panel F shows the percent overlap of the cyclicensemble with 90% of the linear ensemble. Panel G of FIG. 9 shows thepercent overlap of the fibril ensemble with 90% of the linear ensemble.The numeric overlapping percentages are shown in Table 6. Further, PanelH shows the percent overlap of the linear peptide ensemble inside acertain percent of the cyclic peptide ensemble, as that percentage isvaried from 0% to 100%. Note that when the percentage is 90%, theoverlap percentage is equivalent to the converged number in Panel E andTable 6 (7%). This again determines the likelihood that the linearpeptide will adopt a cyclic-like conformation. Panel I shows thecorrelation coefficient between the linear and cyclic distributions, asdefined by first finding the parts of the distributions having densitygreater than a cutoff value, such that a given percentage of the totaldistributions are encompassed, e.g. a density cutoff for the cyclic andlinear distributions that give 60% of the total distributions. Then forthese subdistributions, the correlation coefficient is defined as∫f(τ)g(τ)dτ/√{square root over (∫f(τ)²dτ)} √{square root over(∫g(τ)²dτ)}. Thus defined, the correlation coefficient between thelinear and cyclic distributions converges to about 7% when 100% of therespective distributions are included.

For example, FIG. 9 Panels D-G illustrate the convergence of theensemble overlap values. FIG. 9D shows that the linear and fibrilensembles have an overlap that has converged to less than 0.04%. FIG. 9Eshows that the linear ensemble overlaps with the cyclic ensemble by aconverged value of about 7%. FIG. 9F shows that the cyclic ensembleoverlaps with the linear ensemble by a converged value of 32%. FIG. 9Gshows that the fibril ensemble overlaps with the linear ensemble by aconverged value of about 0.6%.

FIG. 9 Panel H shows the percent overlap of the linear peptide ensembleinside a certain percent of the cyclic peptide ensemble, as thatpercentage is varied from 0% to 100%. Note that when the percentage is90%, the overlap percentage is equivalent to the converged number inPanel E and Table 6 (7%). This again determines the likelihood that thelinear peptide will adopt a cyclic-like conformation.

FIG. 9 Panel I shows the correlation coefficient between the linear andcyclic distributions, as defined by first finding the parts of thedistributions having density greater than a cutoff value, such that agiven percentage of the total distributions are encompassed, e.g. adensity cutoff for the cyclic and linear distributions that give 60% ofthe total distributions. Then for these subdistributions, thecorrelation coefficient is defined as ∫f(τ)g(τ)dτ/√{square root over(∫f(τ)²dτ)} √{square root over (∫g(τ)²dτ)}. Thus defined, thecorrelation coefficient between the linear and cyclic distributionsconverges to about 7% when 100% of the respective distributions areincluded.

FIG. 9 Panel J examines the effects of single residue deletions on thestructural overlap of the linear ensemble with the 90% cyclic ensemble.If a single amino acid confers conformational selectivity, then removingit from the structural alignment will result in a significantly higheroverlap between the distributions. By this test, K16 stands out asconferring the most conformational selectivity to the cyclic peptide.

Two views of the most-representative conformation of HHQK (SEQ ID NO: 1)from the cyclic peptide ensemble, constituting the centroid of thelargest cluster from the cyclic peptide ensemble of structures, areshown in FIG. 7 Panel A in black. As well, the most-representativeconformation in the linear peptide ensemble, constituting the centroidof the largest cluster, is shown in white in FIG. 7, optimallysuperimposed on the cyclic peptide shown in black by aligning them usingRMSD, to make explicit their different orientations. FIG. 7 Panel Bshows the corresponding centroid conformations for the cyclic peptideand linear peptide for the full sequence CGHHQKG (SEQ ID No: 2), againoptimally superimposed by aligning with respect to RMSD.

Table 7 lists values of the Ramachandran backbone and side chaindihedral angles occupied by 13H, 14H, 15Q, and 16K in the centroidstructure of the cyclic peptide ensemble, the centroid structure of thelinear peptide ensemble, and the centroid structure of the fibrilensemble; cyclic and linear centroid conformations are plotted in FIG.7. The centroid structures exhibit several dihedral angles that aresubstantially different between the cyclic conformation and eitherlinear or fibril conformations. Column 1 of Table 7 gives the residueand dihedral angle of interest, column 2 gives the value of the dihedralangle in the centroid structure of the cyclic ensemble, column 3 givesthe value of the dihedral in the linear ensemble centroid, column 4gives the value of the dihedral in the fibril ensemble centroid. It isapparent that many of the cyclic dihedral angles are significantlydifferent then the corresponding dihedral angles in the linear or fibrilcentroids. For example, dihedral C-CA-CB-CG in residue 16K shows adifference of 110 degrees between the cyclic and linear, and 111 degreesbetween the cyclic and fibril. Note that the dihedral angles of thecentroid structures need not be the same as the peak values of thedihedral distributions.

TABLE 7 Dihedral angles in the centroid structures of the linear,cyclic, and fibril ensembles. cyclic Linear 2M4J 13H C-N-CA-C(phi) −70−60 −150 C-N-CA-C(psi) −45 −42 148 C-CA-CB-CG 62 −69 −178 C-CA-N-HN 105100 98 CA-CB-CG-CD2 −109 168 121 CB-CG-ND1-CE1 −176 −172 175CG-CD2-NE2-CE1 −1 −7 −1 HD2-CD2-CG-CB 4 4 12 HE1-CE1-ND1-CG −170 157−180 HE1-CE1-NE2-CD2 170 −156 −179 O-C-CA-CB −100 −89 75 14HC-N-CA-C(phi) −153 −62 −162 C-N-CA-C(psi) 22 −28 137 C-CA-CB-CG 78 166176 C-CA-N-HN 20 115 68 CA-CB-CG-CD2 −130 −13 −21 CB-CG-ND1-CE1 −178 180175 CG-CD2-NE2-CE1 3 1 2 HA-CA-CB-CG −35 46 57 HD2-CD2-CG-CB −2 11 −4HE1-CE1-ND1-CG −167 178 175 HE1-CE1-NE2-CD2 161 179 −176 O-C-CA-CB −31−85 −34 15Q C-N-CA-C(phi) −143 −91 −151 C-N-CA-C(psi) 134 −15 146C-CA-CB-CG −69 −164 −72 C-CA-N-HN 38 90 132 CA-CB-CG-CD −80 −178 −97CG-CD-NE2-1HE2 4 −6 1 NE2-CD-CG-CB −74 130 −74 O-C-CA-CB 78 −77 86 16KC-N-CA-C(phi) −55 −113 −131 C-N-CA-C(psi) 150 −8 104 C-CA-CB-CG −70 −180179 C-CA-N-HN 131 78 91 CA-CB-CG-CD −161 174 171 CD-CE-NZ-HZ1 153 −51 64CE-CD-CG-CB 166 −177 161 CG-CD-CE-HE1 −56 −62 177 CG-CD-CE-HE2 63 70 −51CG-CD-CE-NZ −172 −177 65 O-C-CA-CB 88 −72 62

FIG. 8 again shows the centroid structures for the cyclic, linear, and2M4J fibril ensembles, now using a surface area representation forresidues HHQK (SEQ ID NO: 1). The surface area profile, which would bepresented to an antibody, is different between the centroidconformations. FIG. 8B shows the aligned cyclic and fibril SASA surfacesof HHQK (SEQ ID NO: 1) (left), as well as the aligned cyclic and linearpeptide SASA surfaces of HHQK (SEQ ID NO: 1)(right). Panel 8C Shows thefibril SASA surface of HHQK (SEQ ID NO: 1) by itself, indicating theextent of the burial of the corresponding residues. Thus antibodiesraised to this region in a linear peptide of A-beta will be unlikely tobind cyclic HHQK (SEQ ID NO: 1) (e.g. equally or with similarselectivity), and conversely, antibodies raised to cyclic HHQK (SEQ IDNO: 1) will be unlikely to bind (e.g. equally or with similarselectivity) this region in A-beta.

FIG. 10 shows that the cyclic ensemble does not overlap significantlywith any of the other strains of A-beta fibril. Specifically, theoverlap between the cyclic peptide ensembles distribution and fibrildistributions is zero. FIG. 10A shows the result for PDB 2MXU (2separate views), FIG. 10B for PDB 2LMP, and FIG. 10C for PDB 2LMN (2separate views).

Example 3 Cyclic Compound Construction Comprising a ConformationallyConstrained Epitope

Peptides comprising HHQK (SEQ ID NO: 1) such as Cyclo(CGHHQKG) (SEQ IDNO:2) can be cyclized head to tail.

A linear peptide comprising HHQK (SEQ ID NO:1) and a linker, preferablycomprising 2, 3, or 4 amino acids and/or PEG units, can be synthesizedusing known methods such as Fmoc based solid phase peptide synthesisalone or in combination with other methods. PEG molecules can be coupledto amine groups at the N terminus for example using coupling chemistriesdescribed in Hamley 2014 [6] and Roberts et al 2012 [7], eachincorporated herein by reference. The linear peptide compound may becyclized by covalently bonding 1) the amino terminus and the carboxyterminus of the peptide+linker to form a peptide bond (e.g. cyclizingthe backbone), 2) the amino or carboxy terminus with a side chain in thepeptide+linker or 3) two side chains in the peptide+linker.

The bonds in the cyclic compound may be all regular peptide bonds(homodetic cyclic peptide) or include other types of bonds such asester, ether, amide or disulfide linkages (heterodetic cyclic peptide).

Peptides may be cyclized by oxidation of thiol- or mercaptan-containingresidues at the N-terminus or C-terminus, or internal to the peptide,including for example cysteine and homocysteine. For example twocysteine residues flanking the peptide may be oxidized to form adisulphide bond. Oxidative reagents that may be employed include, forexample, oxygen (air), dimethyl sulphoxide, oxidized glutathione,cystine, copper (II) chloride, potassium ferricyanide, thallium(III)trifluro acetate, or other oxidative reagents such as may be known tothose of skill in the art and used with such methods as are known tothose of skill in the art.

Methods and compositions related to cyclic peptide synthesis aredescribed in US Patent Publication 2009/0215172. US Patent publication2010/0240865, US Patent Publication 2010/0137559, and U.S. Pat. No.7,569,541 describe various methods for cyclization. Other examples aredescribed in PCT Publication WO01/92466, and Andreu et al., 1994.Methods in Molecular Biology 35:91-169.

More specifically, a cyclic peptide comprising the HHQK (SEQ ID NO: 1)epitope can be constructed by adding a linker comprising a spacer withcysteine residues flanking and/or inserted in the spacer. The peptidecan be structured into a cyclic conformation by creating a disulfidelinkage between the non-native cysteines residues added to the N- andC-termini of the peptide. It can also be synthesized into a cycliccompound by forming a peptide bond between the N- and C-termini aminoacids (e.g. head to tail cyclization).

Peptide synthesis is performed by CPC Scientific Inc. (Sunnyvale Calif.,USA) following standard manufacturing procedures.

For example Cyclo(CGHHQKGC) (SEQ ID NO: 13) cyclic peptide comprisingthe conformational epitope HHQK (SEQ ID NO: 1) is constructed in aconstrained cyclic conformation using a disulfide linkage betweencysteine residues added to the N- and C-termini of a peptide comprisingHHQK (SEQ ID NO:1). Two non-native cysteine residues were added to GHHQK(SEQ ID NO: 11) one at the C-terminus and one at the N-terminus. The twocysteines are oxidized under controlled conditions to form a disulfidebridge or reacted head to tail to produce a peptide bond.

As described above, the structure of the cyclic peptide was designed tomimic the conformation and orientation of the amino acid backbone andside chains of HHQK (SEQ ID NO: 1) in A-beta oligomer.

Cyclo(CGHHQKG) (SEQ ID NO: 2)

Cyclo(CGHHQKG) (SEQ ID NO: 2) was synthesized using the following method(CPC Scientific Inc, Sunnyvale Calif.). The protected linear peptide wassynthesized by standard conventional Fmoc-based solid-phase peptidesynthesis on 2-chlorotrityl chloride resin, followed by cleavage fromthe resin with 30% HFIP/DCM. Protected linear peptide was cyclized tothe corresponding protected cyclic peptide by using EDC. HCl/HOBt/DIEAin DMF at low concentration. The protected cyclic peptide wasdeprotected by TFA to give crude cyclic peptide and the crude peptidewas purified by RP HPLC to give pure cyclic peptide after lyophilize.

Cyclo(CGHHQKG) (SEQ ID NO: 2) can be prepared by amide condensation ofthe linear peptide CGHHQKG (SEQ ID NO: 2).

Cyclo(C-PEG2-HHQKG) (SEQ ID NO: 3) can be prepared by amide condensationof the linear compound C-PEG2-HHQKG (SEQ ID NO: 3).

Cyclo(CGHHQK-PEG2) (SEQ ID NO: 4) can be prepared by amide condensationof the linear compound CGHHQK-PEG2 (SEQ ID NO: 4).

Linear (CGHHQKG) (SEQ ID NO: 2) was prepared (CPC Scientific Inc,Sunnyvale Calif.) The protected linear peptide was synthesized bystandard conventional Fmoc-based solid-phase peptide synthesis onFmoc-Gly-Wang resin, then the protected peptide was cleaved by TFA togive crudepeptide and the crude peptide was purified by RP HPLC to givepure peptide after lyophilize, and which was used to conjugate BSA.

Immunogen Construction

The cyclic compound cyclo(CGHHQKG) (SEQ ID NO: 2) was synthesized asdescribed above and then conjugated to BSA and/or KLH (CPC ScientificInc, Sunnyvale Calif.). BSA or KLH was re-activated by SMCC in PBSbuffer, then a solution of the pure peptide in PBS buffer was added tothe conjugation mixture, the conjugation mixture was stirred at r.t for2 h. Then the conjugation mixture was lyophilized after dialysis to givethe conjugation product.

Example 4 Antibody Generation and Selection

A conformational constrained compound optionally a cyclic compound suchas a cyclic peptide comprising HHQK (SEQ ID NO: 1) such ascyclo(CGHHQKG) (SEQ ID NO: 2) peptide is linked to Keyhole LimpetHemocyanin (KLH). The cyclopeptide cyclo(CGHHQKG) (SEQ ID NO: 2) wasmade as described and was sent for mouse monoclonal antibody production(ImmunoPrecise Antibodies LTD (Victoria BC, Canada), following protocolsapproved by the Canadian Council on Animal Care. Mouse sera werescreened using the conformational peptide used for producing theantibodies but can also be screened using a related peptide e.g.cyclo(CGHHQK-PEG2)—peptide (SEQ ID NO: 4), linked to BSA.

Hybridomas were made using an immunogen comprising cyclo(CGHHQKG) (SEQID NO: 2) as further described in Example 6. Hybridoma supernatants werescreened by ELISA and SPR for preferential binding to cyclo(CGHHQKG)(SEQ ID NO: 2) peptide vs linear (unstructured) peptide as describedherein. Positive IgG-secreting clones are subjected to large-scaleproduction and further purification using Protein G.

Example 5 Assessing Binding or Lack Thereof to Plaques/Fibrils

For immunostaining, antibodies described herein, positive control 6E10(1 μg/ml) and isotype controls such as IgG1, IgG2a, and IgG 2b (1 μg/ml,Abcam) are used as primary antibodies. Sections are incubated overnightat 4° C., and washed 3×5 min in TBS-T. Anti-mouse IgG HorseradishPeroxidase conjugated (1:1000, ECL) is applied to sections and incubated45 min, then washed 3×5 min in TBS-T. DAB chromogen reagent (VectorLaboratories, Burlington ON, Canada) is applied and sections rinsed withdistilled water when the desired level of target to background stainingis achieved. Sections are counterstained with Mayer's haematoxylin,dehydrated and cover slips were applied. Slides are examined under alight microscope (Zeiss Axiovert 200M, Carl Zeiss Canada, Toronto ON,Canada) and representative images captured at 50, 200 and 400×magnification using a Leica DC300 digital camera and software (LeicaMicrosystems Canada Inc., Richmond Hill, ON).

Example 6 Methods and Materials Immunogen

Peptides were generated at CPC Scientific, Sunnyvale, Calif., USA (bothcyclic and linear). Peptides were conjugated to KLH (for immunizing) andBSA (for screening) using a trifluoroacetate counter ion protocol.Peptides were desalted and checked by MS and HPLC and deemed 95% pure.Peptides were shipped to IPA for use in production of monoclonalantibodies in mouse.

Antibodies

A number of hybridomas and monoclonal antibodies were generated tocyclo(CGHHQKG) (SEQ ID NO: 2) linked to Keyhole Limpet Hemocyanin (KLH).

Fifty day old female BALB/c mice (Charles River Laboratories, Quebec)were immunized. A series of subcutaneous aqueous injections containingantigen but no adjuvant were given over a period of 19 days. Mice wereimmunized with 100 μg per mouse per injection of a 0.5 mg/mL solution insterile saline of cyclic peptide-KLH. Mice were housed in a ventilatedrack system from Lab Products. All 4 mice were euthanized on Day 19 andlymphocytes were harvested for hybridoma cell line generation.

Fusion/Hybridoma Development

Lymphocytes were isolated and fused with murine SP2/0 myeloma cells inthe presence of poly-ethylene glycol (PEG 1500). Fused cells werecultured using HAT selection. This method uses a semi-solidmethylcellulose-based HAT selective medium to combine the hybridomaselection and cloning into one step. Single cell-derived hybridomas growto form monoclonal colonies on the semi-solid media. 10 days after thefusion event, resulting hybridoma clones were transferred to 96-welltissue culture plates and grown in HT containing medium until mid-loggrowth was reached (5 days).

Hybridoma Analysis (Screening)

Tissue culture supernatants from the hybridomas were tested by indirectELISA on screening antigen (cyclic peptide-BSA) (Primary Screening) andprobed for both IgG and IgM antibodies using a Goatanti-IgG/IgM(H&L)-HRP secondary and developed with TMB substrate.Clones >0.2 OD in this assay were taken to the next round of testing.Positive cultures were retested on screening antigen to confirmsecretion and on an irrelevant antigen (Human Transferrin) to eliminatenon-specific mAbs and rule out false positives. All clones of interestwere isotyped by antibody trapping ELISA to determine if they are IgG orIgM isotype. All clones of interest were also tested by indirect ELISAon other cyclic peptide-BSA conjugates as well as linear peptide-BSAconjugates to evaluate cross-reactivity.

Mouse hybridoma antibodies were screened by indirect ELISA usingcyclo(CGHHQKG) (SEQ ID NO: 2) conjugated to BSA.

ELISA Antibody Screening

Briefly, the ELISA plates were coated with 0.1 ug/wellcyclo(CGHHQKG)—conjugated—BSA (SEQ ID NO: 2) at 100 uL/well in carbonatecoating buffer (pH 9.6) O/N at 4 C and blocked with 3% skim milk powderin PBS for 1 hour at room temperature. Primary Antibody: Hybridomasupernatant at 100 uL/well incubated for 1 hour at 37 C with shaking.Secondary Antibody 1:10,000 Goat anti-mouse IgG/IgM(H+L)-HRP at 100uL/well in PBS-Tween for 1 hour at 37 C with shaking. All washing stepswere performed for 30 mins with PBS-Tween. The substrate3,3′,5,5′-tetramethylbenzidine (TMB) was added at 50 uL/well, developedin the dark and stopped with equal volume 1M HCl.

Positive clones were selected for further testing. Positive clones ofmouse HHQK (SEQ ID NO: 1) hybridomas were tested for reactivity tocyclo(CGHHQKG) (SEQ ID NO: 2) conjugated BSA and human transferrin (HT)by indirect ELISA. Plates were coated with 1) 0.1 ug/wellcyclo(CGHHQKG)—conjugated—BSA (SEQ ID NO: 2) at 100 uL/well in carbonatecoating buffer (pH 9.6) O/N at 4 C; or 2) 0.25 ug/well HT Antigen at 50uL/well in dH2O O/N at 37 C. Primary Antibody: Hybridoma supernatant at100 uL/well incubated for 1 hour at 37 C with shaking. SecondaryAntibody 1:10,000 Goat anti-mouse IgG/IgM(H+L)-HRP at 100 uL/well inPBS-Tween for 1 hour at 37 C with shaking. All washing steps wereperformed for 30 mins with PBS-Tween. The substrate3,3′,5,5′-tetramethylbenzidine (TMB) was added at 50 uL/well, developedin the dark and stopped with equal volume 1M HCl.

ELISA Cyclo Vs Linear CGHHQKG (SEQ ID NO: 2) Compound Selectivity

ELISA plates were coated with 1) 0.1 ug/wellcyclo(CGHHQKG)—conjugated—BSA (SEQ ID NO:2) at 100 uL/well in carbonatecoating buffer (pH 9.6) O/N at 4 C; 2)) 0.1 ug/well linearCGHHQKG—conjugated—BSA (SEQ ID NO:2) at 100 uL/well in carbonate coatingbuffer (pH 9.6) O/N at 4 C; or 3) 0.1 ug/well Negative-Peptide at 100uL/well in carbonate coating buffer (pH 9.6) O/N at 4 C. PrimaryAntibody: Hybridoma supernatant at 100 uL/well incubated for 1 hour at37 C with shaking. Secondary Antibody 1:10,000 Goat anti-mouseIgG/IgM(H+L)-HRP at 100 uL/well in PBS-Tween for 1 hour at 37 C withshaking. All washing steps were performed for 30 mins with PBS-Tween.The substrate TMB was added at 50 uL/well, developed in the dark andstopped with equal volume 1M HCl.

Isotyping

The hybridoma antibodies were isotyped using antibody trap experiments.Trap plates were coated with 1:10,000 Goat anti-mouse IgG/IgM(H&L)antibody at 100 uL/well carbonate coating buffer pH9.6 overnight at 4 C.No blocking step was used. Primary antibody (hybridoma supernatants) wasadded (100 ug/mL). Secondary Antibody 1:5,000 Goat anti-mouse IgGγ-HRPor 1:10,000 Goat anti-mouse IgMμ-HRP at 100 uL/well in PBS-Tween for 1hour at 37 C with shaking. All washing steps were performed for 30 minswith PBS-Tween. The substrate TMB was added at 50 uL/well, developed inthe dark and stopped with equal volume 1M HCl.

SPR Binding Assays—Primary and Secondary Screens SPR Analysis ofAntibody Binding to A-Beta Monomers and Oligomers

A-Beta Monomer and Oligomer Preparation

Recombinant A-beta40 and 42 peptides (California Peptide, Salt Lake CityUtah, USA) were dissolved in ice-cold hexafluoroisopropanol (HFIP). TheHFIP was removed by evaporation overnight and dried in a SpeedVaccentrifuge. To prepare monomers, the peptide film was reconstituted inDMSO to 5 mM, diluted further to 100 μM in dH2O and used immediately.Oligomers were prepared by diluting the 5 mM DMSO peptide solution inphenol red-free F12 medium (Life Technologies Inc., Burlington ON,Canada) to a final concentration of 100 μM and incubated for 24 hours to7 days at 4° C.

SPR Analysis

All SPR measurements were performed using a Molecular Affinity ScreeningSystem (MASS-1) (Sierra Sensors GmbH, Hamburg, Germany), an analyticalbiosensor that employs high intensity laser light and high speed opticalscanning to monitor binding interactions in real time. The primaryscreening of tissue culture supernatants was performed using an SPRdirect binding assay, whereby BSA-conjugated peptides, A-beta42 Monomerand A-beta42 Oligomer are covalently immobilized on individual flowcells of a High Amine Capacity (HAC) sensorchip (Sierra Sensors GmbH,Hamburg, Germany) and antibodies flowed over the surface. Protein Gpurified mAbs were analyzed in a secondary screen using an SPR indirect(capture) binding assay, whereby the antibodies were captured on aprotein A-derivatized sensorchip (XanTec Bioanalytics GmbH, Duesseldorf,Germany) and A-beta40 Monomer, A-beta42 Oligomer, soluble brain extractsand cerebrospinal fluid flowed over the surface. The specificity of theantibodies was verified in an SPR direct binding assay by covalentlyimmobilizing A-beta42 Monomer and A-beta42 Oligomer on individual flowcells of a HAC sensorchip and flowing purified mAbs.

SPR Analysis of Soluble Brain Extracts and CSF Samples

Soluble Brain Extract and CSF Preparation

Human brain tissues and CSFs were obtained from patients assessed at theUBC Alzheimer's and Related Disorders Clinic. Clinical diagnosis ofprobable AD is based on NINCDS-ADRDA criteria [5]. CSFs are collected inpolypropylene tubes, processed, aliquoted into 100 μL polypropylenevials, and stored at −80° C. within 1 hour after lumbar puncture.

Homogenization:

Human brain tissue samples were weighed and subsequently submersed in avolume of fresh, ice cold TBS (supplemented with EDTA-free proteaseinhibitor cocktail from Roche Diagnostics, Laval QC, Canada) such thatthe final concentration of brain tissue is 20% (w/v). Tissue ishomogenized in this buffer using a mechanical probe homogenizer (3×30sec pulses with 30 sec pauses in between, all performed on ice). TBShomogenized samples are then subjected to ultracentrifugation (70,000×gfor 90 min). Supernatants are collected, aliquoted and stored at −80° C.The protein concentration of TBS homogenates is determined using a BCAprotein assay (Pierce Biotechnology Inc, Rockford Ill., USA).

SPR Analysis

Brain extracts from 4 AD patients and 4 age-matched controls, and CSFsamples from 9 AD patients and 9 age-matched controls were pooled andanalyzed. Purified mAbs were captured on separate flow cells of aprotein A-derivatized sensor chip and diluted samples injected over thesurfaces for 180 seconds, followed by 120 seconds of dissociation inbuffer and surface regeneration. Binding responses weredouble-referenced by subtraction of mouse control IgG reference surfacebinding and assay buffer, and the different groups of samples compared

Assessing Binding or Lack Thereof to A-Beta Monomers

In the primary screen of tissue culture supernatants, A-beta42 monomersand A-beta42 oligomers were used in a direct binding assay. In thesecondary screen, A-beta40 monomers and A-beta42 oligomers soluble brainextracts and CSF samples were used in an indirect (capture) bindingassay.

Primary Screen

Tissue culture supernatants were screened for the presence of antibodybinding against their cognate cyclic peptide. Each sample was dilutedand injected in duplicate over the immobilized peptide and BSA referencesurfaces for 120 seconds, followed by injection of running buffer onlyfor a 300-second dissociation phase. After every analytical cycle, thesensor chip surfaces were regenerated. Sensorgrams weredouble-referenced by subtracting out binding from the BSA referencesurfaces and blank running buffer injections, and binding responsereport points collected in the dissociation phase.

Oligomer Binding Assay

Next synthetic A-beta 42 oligomers were generated and immobilized asabove, antibody binding responses analyzed. Antibody binding responsesto A-beta 42 oligomers were compared to binding responses to cyclic.

Verifying Binding to A-Beta Oligomers.

To further verify and validate A-beta42 Oligomer binding, antibodieswere covalently immobilized, followed by the injection over the surfaceof commercially-prepared stable A-beta42 Oligomers (SynAging SAS,Vandoeuvre-{grave over (l)}es-Nancy, France).

Results

ELISA testing found that the majority of hybridoma clones bound thecyclopeptide.

Next clones were tested by ELISA for their binding selectivity forcyclo- and linear—HHQK (SEQ ID NO: 1) compounds. A number of clonespreferentially bound cyclo(CGHHQKG)—conjugated—BSA (SEQ ID NO: 2)compared to linear CGHHQKG—conjugated—BSA (SEQ ID NO: 2).

Isotyping revealed that the majority of clones were IgG including IgG1,IgG2a, and IgG3 clones. Several IgM and IgA clones were also identified,but not pursued further.

A direct binding analysis using surface plasmon resonance was performedto screen for antibodies in tissue culture supernatants that bind to thecyclic peptide of SEQ ID NO: 2. Results are shown in FIG. 11 and Table8.

FIG. 12 plots the correlation between the SPR direct binding assay andthe ELISA results and shows that there is a correlation between thedirect binding and ELISA results.

Clones were retested for their ability to bind cyclic peptide, linearpeptide, A-beta 1-42 monomer and A-beta 1-42 oligomers prepared asdescribed above. Binding assays were performed using SPR as describedabove (Direct binding assays). A number of clones were selected based onthe binding assays performed as shown in Table 8.

The selected clones were IgG mAb. Negative numbers in the primary screenare indicative of no binding (e.g. less than isotype control).

TABLE 8 301 Cyclic- Linear- A β 42 A β 42 Peptide (RU) Peptide (RU)Monomer (RU) Oligomer (RU) 1A5 691 -4.3 20.7 81.2 1C6 193,7 79.2 5.699.9 1D6 463.9 60.6 −1.8 56.8 1F2 473.7 −6 −26..6 55.4 1F3 444 3 −5.9−73.7 87.7 1G6 412.6 −2.7 −20 108.9 1H4 516 0.2 55.4 101.1 2C4 364.3−6.7 26.7 81 4C5 478.9 15 22.7 81,9 5B9 372.3 19.9 −26.6 75.5 5F9 488210 5 21.6 75.3 5G7 615.4 382..4 24 1 80.7 5G9 419) 14.1 9.9 60,6 6F8647.6 17 27.5 100.8 6G3 360 54.6 19.3 74.3 12B12 57S −19.8 69 77 12G11697.9 1150.4 46 66.8

ELISA Prescreen

The ELISA prescreen of hybridoma supernatants identified clones whichshowed increased binding to the cyclic peptides compared to the linearpeptide. A proportion of the clones were reactive to KLH-epitope linkerpeptide. These were excluded from further investigation. The majority ofthe clones were determined to be of the IgG isotype using the isotypingprocedure described herein.

Direct Binding Measured by Surface Plasmon Resonance—Primary Screen

Using surface plasmon resonance the tissue culture supernatantscontaining antibody clones were tested for direct binding to cyclicpeptide, linear peptide, A-beta oligomer and A-beta monomer.

The results for the primary screen are shown in FIG. 11. Panel A showsbinding to cyclic peptide and to linear peptide (unstructured). Panel Bshows binding to A-beta oligomer and A-beta monomer. A number of theclones have elevated reactivity to the cyclic peptide and all cloneshave minimal or no reactivity to linear peptide. There is a generalselectivity for A-beta oligomer binding. Monomer reactivity is around orbelow 0 for most clones.

For select clones comparative binding profile is shown in FIG. 13. Eachclone is assessed for direct binding using surface plasmon resonanceagainst specific epitope in the context of cyclic peptide (structured),linear peptide (unstructured), A-beta monomer, and A-beta oligomer. Aclone reactive preferentially to unstructured epitope (e.g. linearpeptide) was chosen as control, as indicated by an asterisk.

FIG. 12 plots results of a SPR direct binding assay and ELISA resultsfor clone tissue culture supernatants and shows that there is acorrelation between the direct binding and ELISA results.

Example 7 Secondary Screen Immunohistochemistry

Immunohistochemistry was performed on frozen human brain sections, withno fixation or antigen retrieval. In a humidified chamber, non-specificstaining was blocked by incubation with serum-free protein blockingreagent (Dako Canada Inc., Mississauga, ON, Canada) for 1 h. Thefollowing primary antibodies were used for immunostaining: mousemonoclonal isotype controls IgG1, IgG2a, and IgG2b, and anti-amyloidp6E10, all purchased from Biolegend, and selected purified clonesreactive to the cyclopeptide. All antibodies were used at 1 μg/mL.Sections were incubated at room temperature for 1 h, and washed 3×5 minin TBS-T. Anti-Mouse IgG Horseradish Peroxidase conjugated (1:1000, ECL)was applied to sections and incubated 45 min, then washed 3×5 min inTBS-T. DAB chromogen reagent (Vector Laboratories, Burlington ON,Canada) was applied and sections rinsed with distilled water when thedesired level of target to background staining was achieved. Sectionswere counterstained with Mayer's haematoxylin, dehydrated and coverslips were applied. Slides were examined under a light microscope (ZeissAxiovert 200M, Carl Zeiss Canada, Toronto ON, Canada) and representativeimages captured at 20 and 40× magnification using a Leica DC300 digitalcamera and software (Leica Microsystems Canada Inc., Richmond Hill, ON).Images were optimized in Adobe Photoshop using Levels Auto Correction.

CSF and Brain Extracts

Human brain tissues were obtained from the University of Maryland Brainand Tissue Bank upon approval from the UBC Clinical Research EthicsBoard (C04-0595). CSFs were obtained from patients assessed at the UBCHospital Clinic for Alzheimer's and Related Disorders. The study wasapproved by the UBC Clinical Research Ethics Board, and written consentfrom the participant or legal next of kin was obtained prior tocollection of CSF samples. Clinical diagnosis of probable AD was basedon NINCDS-ADRDA criteria. CSFs were collected in polypropylene tubes,processed, aliquoted into 100 μL polypropylene vials, and stored at −80°C. within 1 hour after lumbar puncture.

Homogenization:

Human brain tissue samples were weighed and subsequently submersed in avolume of fresh, ice cold TBS and EDTA-free protease inhibitor cocktailfrom Roche Diagnostics (Laval QC, Canada) such that the finalconcentration of brain tissue was 20% (w/v). Tissue was homogenized inthis buffer using a mechanical probe homogenizer (3×30 sec pulses with30 sec pauses in between, all performed on ice). TBS homogenized sampleswere then subjected to ultracentrifugation (70,000×g for 90 min).Supernatants were collected, aliquoted and stored at −80° C. The proteinconcentration of TBS homogenates was determined using a BCA proteinassay (Pierce Biotechnology Inc, Rockford Ill., USA).

CSF: CSF was pooled from 9 donors with AD and 9 donors without AD.Samples were analyzed by SPR using purified IgG at a concentration of 30micrograms/ml for all antibodies. Mouse IgG was used as an antibodycontrol, and all experiments were repeated at least 2 times.

Positive binding in CSF and brain extracts was confirmed using antibody6E10.

SPR Analysis:

4 brain extracts from AD patients and 4 brain extracts from age-matchedcontrols were pooled and analyzed. Brain samples, homogenized in TBS,included frontal cortex Brodmann area 9. All experiments were performedusing a Molecular Affinity Screening System (MASS-1) (Sierra SensorsGmbH, Hamburg, Germany), an analytical biosensor that employs highintensity laser light and high speed optical scanning to monitor bindinginteractions in real time as described in Example 6. Purified antibodiesgenerated for cyclopeptides described herein were captured on separateflow cells of a protein A-derivatized sensor chip and diluted samplesinjected over the surfaces for 180 seconds, followed by 120 seconds ofdissociation in buffer and surface regeneration. Binding responses weredouble-referenced by subtraction of mouse control IgG reference surfacebinding and assay buffer, and the different groups of samples compared.

Results CSF Brain Extracts and Immunohistochemistry

Several clones were tested for their ability to bind A-beta in CSF,soluble brain extracts and tissue samples of cavaderic AD brains areshown in Table 9. Strength of positivity in Table 9 is shown by thenumber plus signs.

Table 9 and Table 10 provide data for selected clone's bindingselectivity for oligomers over monomer measured as described herein bySPR.

IHC results are also summarized in Table 9 where “+/−” denotes stainingsimilar to or distinct from isotype control but without clear plaquemorphology.

FIG. 14 shows an example of the lack of plaque staining on fresh frozensections with clone 301-17 (12G11) compared to the positive plaquestaining seen with 6E10 antibody.

FIG. 15 shows, antibodies raised to the cyclopeptide comprising HHQK(SEQ ID NO: 1) bound A-beta oligomer preferentially over monomer andalso preferentially bound A-beta in brain extracts and/or CSF of ADpatients.

As shown in Tables 9, 10 and FIGS. 14 and 15, many antibodies raised tothe cyclopeptide comprising HHQK (SEQ ID NO: 1) bound to A-beta in brainextracts and/or CSF, but did not appreciably bind to monomers on SPR,and did not appreciably bind to plaque fibrils by IHC.

TABLE 9 Summary of binding characteristics TABLE 6 Oligomers/ CSF BrainExtract IHC - Plaque Clone # Monomers AD/Non-AD AD/Non-AD Stainingcyclo(CGHHQKG) 301-1D6 (03) +++ + ++ +/− (SEQ ID NO: 2) 301-1F3 (05) + +++ − 301-1H4 (07) +++ + ++ +/− 301-12G11 ++ + ++ − (17) *Scoring isrelative to other clones in the same sample category.

TABLE 10 A-beta Oligomer binding RU values subtracted for monomerbinding Clone tested 301-1D6 (03) RU 22.6

Example 8 Synthetic Oligomer Binding

Serial 2-fold dilutions (7.8 nM to 2000 nM) of commercially-preparedsynthetic amyloid beta oligomers (SynAging SAS, Vandoeuvre-lès-Nancy,were tested for binding to covalently immobilized antibodies. Resultsfor control antibody mAb6E10 is shown in FIG. 16A and mouse control IgGis shown in FIG. 16B. FIG. 16C shows results using an antibody raisedagainst cyclo(CGHHQKG) (SEQ ID NO: 2).

Example 9 Immunohistochemistry on Formalin Fixed Tissues

Human brain tissue was assessed using antibodies raised to cyclo CGHHQKG(SEQ ID NO: 2. The patient had been previously characterized anddiagnosed with Alzheimer's disease with a tripartite approach: (i)Bielschowsky silver method to demonstrate senile plaques andneurofibrillary tangles, (ii) Congo red to demonstrate amyloid and (iii)tau immunohistochemistry to demonstrate tangles and to confirm thesenile plaques are “neuritic”. This tissue was used to test plaquereactivity of selected monoclonal antibody clones. The brain tissueswere fixed in 10% buffered formalin for several days and paraffinprocessed in the Sakura VIP tissue processors. Tissue sections wereprobed with 1 μg/ml of antibody with and without microwave antigenretrieval (AR). The pan-amyloid beta reactive antibody 6E10 was includedalong with selected antibody clones as a positive control. Antibodieswere diluted in Antibody Diluent (Ventana), color was developed withOptiView DAB (Ventana). The staining was performed on the VentanaBenchmark XT IHC stainer. Images were obtained with an Olympus BX45microscope. Images were analyzed blind by a professional pathologistwith expertise in neuropathology.

As shown in Table 11 below, using fixed tissue, the tested antibodieswere negative for specific staining of senile plaque amyloid with orwithout antigen retrieval. 6E10 was used as the positive control.

TABLE 11 Convincing evidence of specific staining of senile plaqueamyloid Epitope Antibodies to test Without AR Plus AR 301 11 Neg Neg 17Neg Neg Positive Control 6E10 strongly positive strongly positive

Example 10 Inhibition of Oligomer Propagation

The biological functionality of antibodies was tested in vitro byexamining their effects on Amyloid Beta (Aβ) aggregation using theThioflavin T (ThT) binding assay. Aβ aggregation is induced by andpropagated through nuclei of preformed small Aβ oligomers, and thecomplete process from monomeric Aβ to soluble oligomers to insolublefibrils is accompanied by concomitantly increasing beta sheet formation.This can be monitored by ThT, a benzothiazole salt, whose excitation andemission maxima shifts from 385 to 450 nm and from 445 to 482 nmrespectively when bound to beta sheet-rich structures and resulting inincreased fluorescence. Briefly, Aβ 1-42 (Bachem Americas Inc.,Torrance, Calif.) was solubilized, sonicated, diluted in Tris-EDTAbuffer (pH7.4) and added to wells of a black 96-well microtitre plate(Greiner Bio-One, Monroe, N.C.) to which equal volumes of cyclopeptideraised antibody or irrelevant mouse IgG antibody isotype controls wereadded, resulting in a 1:5 molar ratio of Aβ1-42 peptide to antibody. ThTwas added and plates incubated at room temperature for 24 hours, withThT fluorescence measurements (excitation at 440 nm, emission at 486 nm)recorded every hour using a Wallac Victor3v 1420 Multilabel Counter(PerkinElmer, Waltham, Mass.). Fluorescent readings from backgroundbuffer were subtracted from all wells, and readings from antibody onlywells were further subtracted from the corresponding wells. As shown inFIG. 17, Aβ42 aggregation, as monitored by ThT fluorescence,demonstrated a sigmoidal shape characterized by an initial lag phasewith minimal fluorescence, an exponential phase with a rapid increase influorescence and finally a plateau phase during which the Aβ molecularspecies are at equilibrium and during which there is no increase influorescence. Co-incubation of Aβ42 with an irrelevant mouse antibodydid not have any significant effect on the aggregation process. Incontrast, co-incubation of Aβ42 with the test antibodies completelyinhibited all phases of the aggregation process. Results obtained withantibody clone 17 (12G11; IgG3 isotype) are shown in FIG. 17. As the ThTaggregation assay mimics the in vivo biophysical/biochemical stages ofAβ propagation and aggregation from monomers, oligomers, protofibrilsand fibrils that is pivotal in AD pathogenesis, the antibodies raised tocyclo CGHHQKG (SEQ ID NO: 2) demonstrate the potential to completelyabrogate this process. Isotype control performed using mouse IgG controlantibody showed no inhibition.

Example 11

Achieving the optimal profile for Alzheimer's immunotherapy: Rationalgeneration of antibodies specific for toxic A-beta oligomers

Objective: Generate antibodies specific for toxic amyloid-β oligomers(AβO)

Background: Current evidence suggests that propagating prion-likestrains of AβO, as opposed to monomers and fibrils, are preferentiallytoxic to neurons and trigger tau pathology in Alzheimer's disease (AD).In addition, dose-limiting adverse effects have been associated with Aβfibril recognition in clinical trials. These observations suggest thatspecific neutralization of toxic AβOs may be desirable for safety andefficacy.

Design/Methods: Computational simulations were employed as describedherein, using molecular dynamics with standardized force-fields toperturb atomic-level structures of Aβ fibrils deposited in the ProteinData Base. It was hypothesized that weakly-stable regions are likely tobe exposed in nascent protofibrils or oligomers. Clustering analysis,curvature, exposure to solvent, solubility, dihedral angle distribution,and Ramachandran angle distributions were all used to characterize theconformational properties of predicted epitopes, which quantifydifferences in the antigenic profile when presented in the context ofthe oligomer vs the monomer or fibril. The candidate peptide epitopeswere synthesized in a cyclic format that may mimic regional AβOconformation, conjugated to a carrier protein, and used to generatemonoclonal antibodies in mice. Purified antibodies were screened by SPRand immunohistochemistry.

Results:

Sixty-six IgG clones against 5 predicted epitopes were selected forpurification based on their ability to recognize the cognate structuredpeptide and synthetic AβO, with little or no binding to unstructuredpeptide, linker peptide, or Aβ monomers. Additional screening identifiedantibodies that preferentially bound to native soluble AβO in CSF andbrain extracts of AD patients compared to controls. Immunohistochemicalanalysis of AD brain allowed for selection of antibody clones that donot react with plaque.

Conclusion: Computationally identified AβO epitopes allowed for thegeneration of antibodies with the desired target profile of selectivebinding to native AD AβOs with no significant cross-reactivity tomonomers or fibrils.

Example 12 Toxicity Inhibition Assay

The inhibition of toxicity of A-beta42 oligomers by antibodies raised tothe cyclopeptide can be tested in a rat primary cortical neuron assay.

Antibody and control IgG are each adjusted to a concentration such as 2mg/mL. Various molar ratios of A-beta oligomer and antibody are testedalong with a vehicle control, A-beta oligomer alone and a positivecontrol such as the neuroprotective peptide humanin HNG.

An exemplary set up is shown in Table 12.

Following preincubation for 10 minutes at room temperature, the volumeis adjusted to 840 microlitres with culture medium. The solution isincubated for 5 min at 37 C. The solution is then added directly to theprimary cortical neurons and cells are incubated for 24 h. Cellviability can be determined using the MTT assay.

TABLE 12 AβO/AB AβO AβO AB AB Medium Final volume molar ratio (μL) (μM)(μM) (μL) (μL) (μL) 5/1 1.68 4.2 0.84  12.73 185.6 200 1/1 1.68 4.2 4.20 63.64 134.7 200 1/2 1.68 4.2 8.4  127.27  71.1 200 AβO workingsolution: 2.2 mg/mL - 500 μM CTRL vehicle: 1.68 μL of oligomer buffer +127.3 μL PBS + 711 μL culture medium CTRL AβO: 1.68 μL of AβO + 127.3 μLPBS + 711 μL culture medium 1.68 μL of AβO + 8.4 μL HNG (100 nM final) +127.3 μL PBS + 702.6 μL culture CTRL HNG: medium

This test was conducted using 301 antibody clone 17. The antibody aloneshowed some toxicity at the highest concentration (1/2 oligomer/antibodyratio), likely due to endotoxin contamination of the antibodypreparation, but demonstrated inhibition of A-beta oligomer toxicitywhen added at lower concentrations (1/1 and 5/1 oligomer/antibodyratios) (FIG. 18).

Example 13 In Vivo Toxicity Inhibition Assay

The inhibition of toxicity of A-beta42 oligomers by antibodies raised tothe cyclopeptide can be tested in vivo in mouse behavioral assays.

The antibody and an isotype control are each pre-mixed with A-beta42oligomers at 2 or more different molar ratios prior tointracerebroventricular (ICV) injection into mice. Control groupsinclude mice injected with vehicle alone, oligomers alone, antibodyalone, and a positive control such as the neuroprotective peptidehumanin. Alternatively, the antibodies can be administered systemicallyprior to, during, and/or after ICV injection of the oligomers. Startingapproximately 4-7 days post ICV injection of oligomers, cognition isassessed in behavioral assays of learning and memory such as the mousespatial recognition test (SRT), Y-Maze assay, Morris water maze modeland novel object recognition model (NOR).

The mouse spatial recognition test (SRT) assesses topographical memory,a measure of hippocampal function (SynAging). The model uses atwo-chamber apparatus, in which the chambers differ in shape, patternand color (i.e. topographical difference). The chambers are connected bya clear Plexiglass corridor. Individual mice are first placed in theapparatus for a 5 min exploration phase where access to only one of thechambers is allowed. Mice are then returned to their home cage for 30min and are placed back in the apparatus for a 5 min “choice” phaseduring which they have access to both chambers. Mice with normalcognitive function remember the previously explored chamber and spendmore time in the novel chamber. A discrimination index (DI) iscalculated as follows: DI=(TN−TF)/(TN+TF), in which TN is the amount oftime spent in the novel chamber and TF is the amount of time spent inthe familiar chamber. Toxic A-beta oligomers cause a decrease in DIwhich can be partially rescued by the humanin positive control.Performance of this assay at different time points post ICV injectioncan be used to evaluate the potential of antibodies raised to thecyclopeptide to inhibit A-beta oligomer toxicity in vivo.

The Y-maze assay (SynAging) is a test of spatial working memory which ismainly mediated by the prefrontal cortex (working memory) and thehippocampus (spatial component). Mice are placed in a Y-shaped mazewhere they can explore 2 arms. Mice with intact short-term memory willalternate between the 2 arms in successive trials. Mice injected ICVwith toxic A-beta oligomers are cognitively impaired and show randombehavior with alternation close to a random value of 50% (versus ˜70% innormal animals). This impairment is partially or completely reversed bythe cholinesterase inhibitor donepezil (Aricept) or humanin,respectively. This assay provides another in vivo assessment of theprotective activity of test antibodies against A-beta oligomer toxicity.

The Morris water maze is another widely accepted cognition model,investigating spatial learning and long-term topographical memory,largely dependent on hippocampal function (SynAging). Mice are trainedto find a platform hidden under an opaque water surface in multipletrials. Their learning performance in recalling the platform location isbased on visual clues and video recorded. Their learning speed, which isthe steadily reduced time from their release into the water untilfinding the platform, is measured over multiple days. Cognitively normalmice require less and less time to find the platform on successive days(learning). For analyzing long-term memory, the test is repeatedmultiple days after training: the platform is taken away and the numberof crossings over the former platform location, or the time of the firstcrossing, are used as measures to evaluate long-term memory. Miceinjected ICV with toxic A-beta oligomers show deficits in both learningand long-term memory and provide a model for evaluating the protectiveactivity of test antibodies.

The Novel Object Recognition (NOR) model utilizes the normal behavior ofrodents to investigate novel objects for a significantly longer timethan known objects, largely dependent on perirhinal cortex function(SynAging). Mice or rats are allowed to explore two identical objects inthe acquisition trial. Following a short inter-trial interval, one ofthe objects is replaced by a novel object. The animals are returned tothe arena and the time spent actively exploring each object is recorded.Normal rodents recall the familiar object and will spend significantlymore time exploring the novel object. In contrast, A-betaoligomer-treated rodents exhibit clear cognitive impairment and willspend a similar amount of time investigating both the ‘familiar’ and‘novel’ object. This can be transiently reversed with known clinicalcognitive enhancers (e.g. donepezil). The NOR assay can be performedmultiple times in longitudinal studies to assess the potential cognitivebenefit of test antibodies.

In addition to behavioral assays, brain tissue can be collected andanalyzed for levels of synaptic markers (PSD95, SNAP25, synaptophysin)and inflammation markers (IL-1-beta). Mice are sacrificed at ˜14 dayspost-ICV injection of oligomers and perfused with saline. Hippocampi arecollected, snap frozen and stored at −80° C. until analyzed. Proteinconcentrations of homogenized samples are determined by BCA.Concentration of synaptic markers are determined using ELISA kits(Cloud-Clone Corp, USA). Typically, synaptic markers are reduced by25-30% in mice injected with A-beta oligomers and restored to 90-100% bythe humanin positive control. Concentrations of the IL-1-betainflammatory markers are increased approximately 3-fold in mice injectedwith A-beta oligomers and this increase is largely prevented by humanin.These assays provide another measure of the protective activity of testantibodies at the molecular level.

Example 14 In Vivo Propagation Inhibition Assay

In vivo propagation of A-beta toxic oligomers and associated pathologycan be studied in various rodent models of Alzheimer's disease (AD). Forexample, mice transgenic for human APP (e.g. APP23 mice) or human APPand PSEN1 (APPPS1 mice) express elevated levels of A-beta and exhibitgradual amyloid deposition with age accompanied by inflammation andneuronal damage. Intracerebral inoculation of oligomer-containing brainextracts can significantly accelerate this process 13, 14). These modelsprovide a system to study inhibition of A-beta oligomer propagation bytest antibodies administered intracerebrally or systemically.

Example 15 CDR Sequencing

301-12G11 which was determined to have an IgG3 heavy chain and a kappalight chain was selected for CDR and variable regions of the heavy andlight chains.

RT-PCR was carried out using 5′ RACE and gene specific reverse primerswhich amplify the appropriate mouse immunoglobulin heavy chain(IgG1/IgG3/IgG2A) and light chain (kappa) variable region sequences.

The specific bands were excised and cloned into pCR-Blunt II-TOPO vectorfor sequencing, and the constructs were transformed into E. coli

At least 8 colonies of each chain were picked & PCR screened for thepresence of amplified regions prior to sequencing. Selected PCR positiveclones were sequenced.

The CDR sequences are in Table 13. The consensus DNA sequence andprotein sequences of the variable portion of the heavy and light chainare provided in Table 14.

TABLE 13 Chain CDR Sequence SEQ ID NO. Heavy CDR-H1 GYSFTSYW 22 CDR-H2VHPGRGVST 23 CDR-H3 SRSHGNTYWFFDV 24 Light CDR-L1 QSIVHSNGNTY 25 CDR-L2KVS 26 CDR-L3 FQGSHVPFT 27

TABLE 14Consensus DNA sequence and translated protein sequences of the variable region.The determining regions (CDRs) are underlined according to IMTG/LIGM-DB.complementarity Isotype Consensus DNA Sequence Protein sequence IgG3ATGGGATGGAGCTGTATCATCCTCTTTTTGGTAGCAACAGCTACA MGWSCIILFLVATATGSEQ ID NO: GGTGTCCACTCCCAGGTCCAACTGCAGCAGCCTGGGGCTGAGCTTVHSQVQLQQPGAELVK 28, 29 GTGAAGCCTGGGGCTTCAGTGAAAATGTCCTGCAAGGCTTCT GGCPGASVKMSCKAS GYSF TACAGCTTCACCAGCTACTGG ATAAACTGGGTGAAGCAGAGGCCT TSYWINWVKQRPGQGL GGACAAGGCCTTGAGTGGATTGGAGAT GTTCATCCTGGTAGAGGT EWIGDVHPGRGVST YN GTTTCTACC TACAATGCGAAGTTCAAGAGCAAGGCCACACTGACTAKFKSKATLTLDTSSS CTAGACACGTCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGTAYMQLSSLTSEDSAV ACATCTGAGGACTCTGCGGTCTATTATTGT TCAAGATCCCACGGT YYCSRSHGNTYWFFDV AATACCTACTGGTTCTTCGATGTC TGGGGCGCAGGGACCACGGTCWGAGTTVTVSSATTTA ACCGTCTCCTCAGCTACAACAACAGCCCCATCT PS KappaATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCT MKLPVRLLVLMFWIPASEQ ID NO: GCTTCCAGCAGTGATGTTTTGATGACCCAAACTCCACTCTCCCTGSSSDVLMTQTPLSLPV 30, 31 CCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTSLGDQASISCRSS QSI CAGAGCATTGTACATAGTAATGGAAACACCTAT TTAGAATGGTACVHSNGNTY LEWYLQKP CTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAA AGTTGQSPKLLIY KVS NRFS TCC AACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGAGVPDRFSGSGSGTDFT TCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGLKISRVEAEDLGVYYC GATCTGGGAGTTTATTACTGC TTTCAAGGTTCACATGTTCCATTCFQGSHVPFT FGSGTKL ACG TTCGGCTCGGGGACAAAGTTGGAAATAAAACGGGCTGATGCT EIKRADA

TABLE 15 A-beta Sequences 1) HHQK (SEQ ID NO: 1)CGHHQKG, cyclo(CGHHQKG) (SEQ ID NO: 2)CHHQKG, C-PEG2-HHQKG, cyclo C-PEG2-HHQKG ((SEQ ID NO: 3)CGHHQK, CGHHQK-PEG2, cyclo(CGHHQK)-PEG2 (SEQ ID NO: 4)VHHQ (SEQ ID NO: 5) VHHQKL (SEQ ID NO: 6) HHQKL (SEQ ID NO: 7)GHHQKG (SEQ ID NO: 9) HHQKG (SEQ ID NO: 10) GHHQK (SEQ ID NO: 11)VHHQK (SEQ ID NO: 12) CGHHQKGC (SEQ ID NO: 13) EVHHQK (SEQ ID NO: 18)HQKL (SEQ ID NO: 20) CGHHQKC, cyclo(CGHHQKC) (SEQ ID NO: 17) 2)HQKLVFFAED (SEQ ID NO: 16) HHQKLVFFAEDVGSNK (SEQ ID NO: 19)HQKLV (SEQ ID NO: 21) HHQKLV (SEQ ID NO: 8) HQKLVF (SEQ ID NO: 14)HQKLVFF (SEQ ID NO: 15)

TABLE 16 Human A-beta 1-42 DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA(SEQ ID NO: 32)

While the present application has been described with reference to whatare presently considered to be the preferred examples, it is to beunderstood that the application is not limited to the disclosedexamples. To the contrary, the application is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety. Specifically, the sequences associated with eachaccession numbers provided herein including for example accessionnumbers and/or biomarker sequences (e.g. protein and/or nucleic acid)provided in the Tables or elsewhere, are incorporated by reference inits entirely.

The scope of the claims should not be limited by the preferredembodiments and examples, but should be given the broadestinterpretation consistent with the description as a whole.

CITATIONS FOR REFERENCES REFERRED TO IN THE SPECIFICATION

-   [1] Gabriela A. N. Crespi, Stefan J. Hermans, Michael W. Parker, and    Luke A. Miles. Molecular basis for mid-region amyloid-b capture by    leading Alzheimer's disease immunotherapies SCIENTIFIC REPORTS 5:    9649, 20151 DOI: 10.1038/srep09649-   [2] Vincent J. Hilser and Ernesto Freire. Structure-based    calculation of the equilibrium folding pathway of proteins.    correlation with hydrogen exchange protection factors. J. Mol.    Biol., 262:756-772, 1996. The COREX approach.-   [3] Samuel I. A. Cohen, Sara Linse, Leila M. Luheshi, Erik    Hellstrand, Duncan A. White, Luke Rajah, Daniel E. Otzen, Michele    Vendruscolo, Christopher M. Dobson, and Tuomas P. J. Knowles.    Proliferation of amyloid-β42 aggregates occurs through a secondary    nucleation mechanism. Proc. Natl.l Acad. Sci. USA,    110(24):9758-9763, 2013.-   [4] Pietro Sormanni, Francesco A. Aprile, and Michele Vendruscolo.    The camsol method of rational design of protein mutants with    enhanced solubility. Journal of Molecular Biology, 427(2):478-490,    2015.-   [5] Deborah Blacker, MD, ScD; Marilyn S. Albert, PhD; Susan S.    Bassett, PhD; Rodney C. P. Go, PhD; Lindy E. Harrell, MD, PhD;    Marshai F. Folstein, MD Reliability and Validity of NINCDS-ADRDA    Criteria for Alzheimer's Disease The National Institute of Mental    Health Genetics Initiative. Arch Neurol. 1994; 51(12):1198-1204.    doi:10.1001/archneur.1994.00540240042014.-   [6] Hamley, I. W. PEG-Peptide Conjugates 2014; 15, 1543-1559;    dx.doi.org/10.1021/bm500246w-   [7] Roberts, M J et al Chemistry for peptide and protein PEGylation    64: 116-127.-   [8] J. X. Lu, W. Qiang, W. M. Yau, C. D. Schwieters, S. C.    Meredith, R. Tycko, MOLECULAR STRUCTURE OF BETA-AMYLOID FIBRILS IN    ALZHEIMER'S DISEASE BRAIN TISSUE. CELL Vol. 154 p. 1257 (2013)-   [9] Y. Xiao, B. MA, D. McElheny, S. Parthasarathy, F. Long, M.    Hoshi, R. Nussinov, Y. Ishii, A BETA (1-42) FIBRIL STRUCTURE    ILLUMINATES SELF-RECOGNITION AND REPLICATION OF AMYLOID IN    ALZHEIMER'S DISEASE. NAT. STRUCT. MOL. BIOL. Vol. 22 p. 499 (2015).-   [10] A. Petkova, W. Yau, R. Tycko EXPERIMENTAL CONSTRAINTS ON    QUATERNARY STRUCTURE IN ALZHEIMER'S BETA-AMYLOID FIBRILS    BIOCHEMISTRY V. 45 498 2006.-   [11] Giulian D, Haverkamp L J, Yu J, Karshin W, Tom D, Li J,    Kazanskaia A, Kirkpatrick J, Roher A E. The HHQK domain of β-amyloid    provides a structural basis for the immunopathology of Alzheimer's    disease, J. Biol. Chem. 1998, 273(45), 29719-26.-   [12] Winkler K, Scharnagl H, Tisljar U, HoschOtzky H, Friedrich I,    Hoffmann M M, Hüttinger M, Wieland H, März W. Competition of Aβ    amyloid peptide and apolipoprotein E for receptor-mediated    endocytosis. J. Lipid Res. 1999, 40(3), 447-55.

1. A cyclic compound comprising: an A-beta peptide the peptidecomprising HQK and up to 6 A-beta contiguous residues, and a linker,wherein the linker is covalently coupled to the A-beta peptideN-terminus residue and the A-beta C-terminus residue, optionally whereinthe A-beta peptide is selected from a peptide having a sequence of anyone of SEQ ID NOS: 1-16, optionally selected from HHQK (SEQ ID NO: 1),HQK, HHQKL (SEQ ID NO: 7), VHHQKL (SEQ ID NO: 6), VHHQ (SEQ ID NO: 5),and HQKL (SEQ ID NO:20). 2.-11. (canceled)
 12. An immunogen comprisingthe cyclic compound of claim 1 optionally wherein the compound iscoupled to a carrier protein or immunogenicity enhancing agent,optionally wherein the carrier protein is bovine serum albumin (BSA) orthe immunogenicity-enhancing agent is keyhole Keyhole Limpet Haemocyanin(KLH). 13.-14. (canceled)
 15. A composition comprising the compound ofclaim 1 or an immunogen comprising said compound.
 16. The composition ofclaim 15, further comprising an adjuvant, optionally wherein theadjuvant is aluminum phosphate or aluminum hydroxide.
 17. (canceled) 18.An isolated conformation specific and/or selective antibody thatspecifically and/or selectively binds to an A-beta peptide having asequence of HQK or a related epitope sequence presented in the cycliccompound of claim 1, or an immunogen comprising said cyclic compound,optionally having a sequence of SEQ ID NO: 2, 3, 4 or
 32. 19. (canceled)20. The antibody of claim 18 wherein the A-beta peptide and/or epitopecomprises or consists of HHQK (SEQ ID NO: 1), VHHQKL (SEQ ID NO: 6),VHHQ (SEQ ID NO: 5), and HQKL (SEQ ID NO: 20).
 21. (canceled)
 22. Theantibody of claim 18, wherein the antibody selectively binds A-betaoligomer over A-beta monomer and/or A-beta fibril, optionally whereinthe selectivity is at least 2 fold, at least 3 fold, at least 5 fold, atleast 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, atleast 50 fold, at least 100 fold, at least 500 fold, at least 1000 foldmore selective for A-beta oligomer over A-beta monomer and/or A-betafibril, optionally wherein the antibody lacks or has negligible bindingto A-beta monomer and/or A-beta fibril plaques in situ. 23.-25.(canceled)
 26. The antibody of claim 18 wherein the antibody is amonoclonal antibody or a polyclonal antibody or a humanized antibody,and/or therein the antibody is an antibody binding fragment selectedfrom Fab, Fab′, F(ab′)2, scFv, dsFv, ds-scFv, dimers, nanobodies,minibodies, diabodies, and multimers thereof. 27.-28. (canceled)
 29. Theantibody of claim 18, comprising a light chain variable region and aheavy chain variable region, optionally fused, the heavy chain variableregion comprising complementarity determining regions CDR-H1, CDR-H2 andCDR-H3, the light chain variable region comprising complementaritydetermining region CDR-L1, CDR-L2 and CDR-L3 and with the amino acidsequences of said CDRs comprising the sequences: (SEQ ID NO: 22) CDR-H1GYSFTSYW (SEQ ID NO: 23) CDR-H2 VHPGRGVST (SEQ ID NO: 24) CDR-H3SRSHGNTYWFFDV (SEQ ID NO: 25) CDR-L1 QSIVHSNGNTY (SEQ ID NO: 26) CDR-L2KVS (SEQ ID NO: 27) CDR-L3 FQGSHVPFT


30. The antibody of claim 18, wherein the antibody comprises a heavychain variable region comprising: i) an amino acid sequence as set forthin SEQ ID NO: 29; ii) an amino acid sequence with at least 50%, at least60%, at least 70%, at least 80% or at least 90% sequence identity to SEQID NO: 29, wherein the CDR sequences are as set forth in SEQ ID NO: 22,23 and 24, or iii) a conservatively substituted amino acid sequence i);and/or wherein the antibody comprises a light chain variable regioncomprising i) an amino acid sequence as set forth in SEQ ID NO: 31, ii)an amino acid sequence with at least 50%, at least 60%, at least 70%, atleast 80%, at least 90% sequence identity to SEQ ID NO: 31, wherein theCDR sequences are as set forth in SEQ ID NO: 25, 26 and 27, or iii) aconservatively substituted amino acid sequence of i).
 31. (canceled) 32.The antibody of claim 18, wherein the heavy chain variable region aminoacid sequence is encoded by a nucleotide sequence as set forth in SEQ IDNO: 28 or a codon degenerate or optimized version thereof; and/or theantibody comprises a light chain variable region amino acid sequenceencoded by a nucleotide sequence as set out in SEQ ID NO: 30 or a codondegenerate or optimized version thereof, optionally wherein the heavychain variable region comprises or consists of an amino acid sequence asset forth in SEQ ID NO: 29 and/or the light chain variable regioncomprises or consists of an amino acid sequence as set forth in SEQ IDNO:
 31. 33.-34. (canceled)
 35. An immunoconjugate comprising theantibody of claim 18 and a detectable label or cytotoxic agent,optionally wherein the detectable label comprises a positron emittingradionuclide, optionally for use in subject imaging such as PET imaging.36. (canceled)
 37. A composition comprising the antibody of claim 18, oran immunoconjugate comprising said antibody, optionally with a diluent.38. A nucleic acid molecule encoding the antibody of claim 18 or aproteinaceous immunoconjugates comprising said antibody or a vectorcomprising said nucleic acid.
 39. (canceled)
 40. A cell expressing theantibody of claim 18, optionally wherein the cell is a hybridoma cell.41. A kit comprising a cyclic compound comprising an A-beta peptide thepeptide comprising HQK and up to 6 A-beta contiguous residues, and alinker, wherein the linker is covalently coupled to the A-beta peptideN-terminus residue and the A-beta C-terminus residue, an immunogencomprising said compound, the antibody of claim 18, an immunoconjugate,or composition comprising said antibody, a nucleic acid molecule orvector encoding said antibody or a cell expressing said antibody.
 42. Amethod of making the antibody of claim 18, comprising administering acyclic compound comprising an A-beta peptide the peptide comprising HQKand up to 6 A-beta contiguous residues, and a linker, wherein the linkeris covalently coupled to the A-beta peptide N-terminus residue and theA-beta C-terminus residue or an immunogen comprising said compound or acomposition comprising said compound or immunogen to a subject andisolating antibody and/or cells expressing antibody specific orselective for the compound or immunogen administered and/or A-betaoligomers, optionally lacking or having negligible binding to a linearpeptide comprising the A-beta peptide and/or lacking or havingnegligible plaque binding.
 43. A method of determining if a biologicalsample comprises A-beta, the method comprising: a. contacting thebiological sample with the antibody of claim 18 or an immunoconjugatecomprising said antibody; and b. detecting the presence of any antibodycomplex, optionally wherein the amount of complex is measured. 44.-49.(canceled)
 50. A method of measuring a level of A-beta in a subject, themethod comprising administering to a subject at risk or suspected ofhaving or having AD, an immunoconjugate comprising the antibody of claim35 wherein the antibody is conjugated to a detectable label, optionallywherein the label is a positron emitting radionuclide; and detecting thelabel, optionally quantitatively detecting the label.
 51. (canceled) 52.A method of inducing an immune response in a subject, comprisingadministering to the subject the compound or combination of compounds ofclaim 1, optionally a cyclic compound comprising HQK or HHQK (SEQ IDNO: 1) or a related epitope peptide sequence, an immunogen and/orcomposition comprising said compound or said immunogen; and optionallyisolating cells and/or antibodies that specifically or selectively bindthe A-beta peptide in the compound or immunogen administered.
 53. Amethod of inhibiting A-beta oligomer propagation, the method comprisingcontacting a cell or tissue expressing A-beta with or administering to asubject in need thereof an effective amount of the A-beta oligomerspecific or selective antibody of claim 18 or an immunoconjugatecomprising said antibody, to inhibit A-beta aggregation and/or oligomerpropagation.
 54. A method of treating AD and/or other A-beta amyloidrelated diseases, the method comprising administering to a subject inneed thereof i) an effective amount of the antibody of claim 18, animmunoconjugate comprising said antibody, or a pharmaceuticalcomposition comprising said antibody; 2) administering an isolatedcyclic compound comprising HQK, HHQK (SEQ ID NO: 1) or a related epitopesequence or immunogen or pharmaceutical composition comprising saidcyclic compound, or 3) a nucleic acid or vector comprising a nucleicacid encoding the antibody of 1) or the cyclic compound or immunogen of2), to a subject in need thereof, optionally wherein the antibody,immunoconjugate, immunogen, composition or nucleic acid or vector isadministered directly to the brain or other portion of the CNS. 55.-58.(canceled)
 59. An isolated peptide comprising an A beta peptideconsisting of the sequence of any one of the sequences set forth in SEQID NO:1-11, 13, 17 or 20 or a nucleic acid molecule encoding saidisolated peptide. 60.-63. (canceled)